Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations.

J. Mol. Biol. (1991) 222, 99-124

Amino Acid Substrate Specificity of Escherichia coli Phenylalanyl-tRNA Synthetase Altered by Distinct Mutations Peter Kast and Hauke Henneckef Mikrobiologisches

Eidgeniissische Technische Hochschule Schmelzbergatra.sse 7 CH-8092 Ziirich, Switzerland Institut,

(Received 21 March 1991; accepted 24 June 1991) Neither the tertiary structure nor the location of active sites are known for phenylalanyltRNA synthetase (PheRS; cQ2 structure), a member of class II aminoacyl-tRNA synthetases. In an attempt to detect the phenylalanine (Phe) binding site, two Escherichia coli PheRS mutant strains (pheS), which were resistant to p-fluorophenylalanine (p-F-Phe) were analysed genetically. The pheS mutations were found to cause Ala294 to Ser294 exchanges in the a subunits from both independent strains. This alteration (S294) resided in the well-conserved C-terminal part of the c( subunit, precisely within motif 3, a typical class II tRNA synthetase sequence. We thus propose that motif 3 participates in the formation of the Phe binding site of PheRS. Mutation 5294 was also the key for proposing a mechanism by which the substrate analogue p-F-Phe is excluded from the enzymatic reaction; this may be achieved by steric interactions between the para-position of the aromatic ring and the amino acid residue at position 294. The Phe binding site model was then tested by replacing the alanine at position 294 as well as the two flanking phenylalanines (positions 293 and 295) by a number of selected other amino acids. In vivo and in vitro results demonstrated that Phe293 and Phe295 are not directly involved in substrate binding, but replacements of those residues affected PheRS stability. However, exchanges at position 294 altered the binding of Phe, and certain mutants showed pronounced changes in specificity towards Phe analogues. Of particular interest was the Gly294 PheRS in which presumably an enlarged cavity for the para position of the aromatic ring allowed an increased aminoacylation of tRNA with p-F-Phe. Moreover, the larger para-chloro and para-bromo derivatives of Phe could interact with this enzyme in vitro and became highly toxic in vivo. The possible exploitation of the Gly294 mutant PheRS for the incorporation of non-proteinogenic amino acids into proteins is discussed. Keywords: aromatic amino acid binding site; oligonucleotide-directed mutagenesis; p-fluoro-phenylalanine resistance; phenylalanyl-tRNA synthetase; pheS gene mutations.

Recently, most aminoacyl-tRNA synthetases were classified into two non-related groups, according to the presence or absence of typical short consensus sequences (Burbaum et al., 1990; Eriani et al., 199Oa). For three class I members, the threedimensional structures derived from X-ray analyses are available: TyrRSS from Bacillus stearothermophilus (Brick et al., 1989), GlnRS from Escherichia coli (Rould et al., 1989) and an active fragment of

1. Introduction By coupling amino acids to their cognate tRNAs, aminoacyl-tRNA synthetases catalyse key reactions in translating the genetic code. Aminoacyl-tRNA synthetases responsible for the same amino acid are generally well conserved across species. However, in spite of their common reaction mechanism, the individual tRNA synthetases for each of the 20 amino acids in a given organism are widely diverse in sequence, subunit size and quarternary structure (for reviews, see Schimmel & 5611, 1979; Schimmel, 1987). To know the reason for this diversity might help unravel the origin of the genetic code (Weiner & Maizels, 1987).

$ Abbreviations used: XxxRS, aminoacyl-tRNA synthetase for amino acid Xxx in 3-letter-code. aa. amino acid(s); bp, base-pair(s); 3-F-Tyr, 3-fluorotyrosine; IPTG, isopropyl-j?-n-thiogalactopyranoside: kb, lo3 base-pairs; p-F-Phe, p-Cl-Phe, m-Cl-Phe and p-Br-Phe, para-fluoro-, para-chloro-, meta-chloro- and para-bromo-phenylalanines; R, resistant; SDS. sodium dodecylsulphate; ts, thermosensitive.

7 Author to whom all correspondence should be addressed. 002%2836/91/21009s-26

$03.00/0

99

0 1991 Academic Press Limited

100

P. Kast and H. Hennecke

a-subunit (326 em)

, lkb

&subunit ’

(766 aa)

, thrS

infC

@T

pheS

pheT

himA ’

EcM

Figure 1. (a) Organization of the PheRS genes. The genes pheS and pheT (encoding PheRS a and jl subunits, respectively) are arranged in an operon at min 37 of the E. coli linkage map (Bachmann, 1990). Besides pheS and pheT the 1925 kb EcoRI/HindIII fragment contains the genes encoding threonyl-tRNA synthetase (thrS), initiation factor IF3 (infc), ribosomal proteins A (or L35; encoded by rpml) and L20 (rpZT), and the N-terminal end of the a subunit of integration host factor (himA) (Fayat et al., 1983; Mechulam et aZ., 1985; Wada & Sako, 1987). PheRS is drawn as suggested by Dessen et al. (1990). (b) Subcloning of pheS genes. The pKSC-X plasmids (Table 1) obtained after cloning of the chromosomal pheST region were digested with SmaI and Hind11 and the 1181 bp blunt-end pheS fragment was ligated to a SmaI-linearized pBluescript KS (+) vector (pBLS). Both insert orientations (pKSBl-X and pKSB2-X) were necessary for single-strand sequencing of pheS on both strands. The pKSBl-X plasmid type was utilized for high expression of pheS from the Zacpromoter (Pr,,). blu, gene for /?-lactamase conferring ampicillin resistance. The -X needs to be replaced by other letters to indicate the source of the pheS gene, as follows: A, AB1360-12; K, KlO-F6; W, wild-type.

E. coli MetRS (Brunie et al., 1990). The structures suggest that these aminoacyl-tRNA synthetases are composed of distinct domains (Schimmel, 1987) that are conserved in secondary and tertiary rather than primary structure (Blow et al., 1983; Rould et al., 1989). In addition, the class I-specific consensus sequence elements were shown to carry the active sites. A HIGH signature sequence participates in the N-terminal section of a structurally conserved nucleotide binding fold (“Rossmann fold”; Rossmann et al., 1974) for the binding of ATP (Blow et al., 1983; Leatherbarrow et al., 1985; Webster et al., 1987; Burbaum et al., 1990). Another sequence (KMSKS) maps near the C-terminal end of the Rossmann fold (Burbaum et al., 1990). It was originally identified by affinity labelling experiments (Hountondji et al., 1985) and is thought to be close to the 3’-end of the bound tRNA substrate (Hountondji et al., 1986; Meinnel et al., 1990). Furthermore, it appears to interact with ATP (Fersht et al., 1988; Rould et al., 1989; Hountondji et al., 1990). To date, the only published crystal structure of a class II tRNA synthetase is that of E. coli SerRS

(Cusack et al., 1990). The characteristic Rossmann fold of class I enzymes was not present in SerRS, which confirmed the partition of tRNA synthetases into two classes also on a conformational basis. The SerRS substrates could not be co-crystallized; therefore, their binding sites have not yet been determined and related to the class II consensus motifs. Our own research focusses on phenylalanyl-tRNA synthetase (PheRS) of E. coli. The two-step reaction

mechanism

of this enzyme

(SSll & Schimmel,

1974; Fasiolo t Fersht, 1978; Baltzinger & Holler, 1982) can be schematically summarized as: E + Phe + ATPe[E*PhewAMP] [E . Phe N AMP] + tRNAPhe= Phe-tRNAPhc + AMP,

+ PP,,

(1) (2)

where E and PP, stand for PheRS and inorganic pyrophosphate, respectively. PheRS is among the largest aminoacyl-tRNA synthetases known and displays an a#* quarternary structure (Fayat et al., 1974; Hanke et al., 1974; Schimmel, 1987). The PheRS genes pheS and pheT (encoding the a and fi subunits, respectively) had been isolated on a 2 transducing phage (Hennecke et al., 1977a) and

Altered Phenylalanyl-tRNA their nucleotide sequences were determined (Fayat et al., 1983; Mechulam et al., 1985). The gene arrangement is shown in Figure l(a). According to the presence of certain consensus sequence elements, PheRS belongs to the less well characterized class II tRNA synthetases (Eriani et al., 199Oa). Neither the tertiary structure of PheRS nor t,he location of the binding sites for Phe or ATP within t,he primary sequence are known. Affinity labelling experiments with E. coli PheRS implied the large /? subunit to contain tRNAPhe binding sites (Khodyreva et al., 1985), and Hountondji et al. (1987) identified sequences in the N-terminal region of the b subunit to be near the binding site of the 3’-end of tRNAPh”. Other substrate cross-linking studies suggested the active site to lie at the a/I interface in PheRS of E. coli (Khodyreva et al., 1985) and yeast (Baltzinger et al., 1979). One aim in this work was to localize the Phe binding s&e of PheRS. For this purpose, we analysed two independently isolated E. coli mutant strains that are resistant to p-fluoro-phenylalanine (p-F-Phe). This phenotype is caused by mutant PheRSs that exclude, in contrast to the wild-type enzyme, this toxic substrate analogue (Richmond, 1962) from the enzymatic reaction (Hennecke & B&k. 1975). It was expected that a p-F-Phe resistance mutation, which leads to a discrimination in PheRS between Phe and the slightly bigger substrate analogue, should point directly to the Phe binding sit,e. In addition, the p-F-PheR mutations appeared particularly suitable to investigate the determinants that, govern substrate specificity. The specificity of an enzyme for a substrate is given by the structural complemrntarity of the binding site to that substrate in its transition state of the enzymatic reaction (Fersht, 19856). Consequently, an exchange of amino acid residues involved in substrate binding may alter the substrate specificity of that enzyme. Experiments of that kind were performed extensively with serine proteases of known tertiary structure such as trypsin (Craik et al., 1985) and subtilisin (E&e11 et al., 1986; Wells et al., 1987a,b). Whilst these studies dealt with enzymes of comparably poor specificity (Bosshard, 1976), an alteration of t,he substrate specificity of the highly specific aminoacyl-tRNA synthetases, which possess efficient edltmg and proofreading mechanisms (Fersht, 19856; Freist, 1989), seems even more challenging. In the present work, we wished to work out the molecular basis of the change in substrate specificity in the p-F-Phe-resistant PheRS. Both independent p-F-Phe resistance mutations were previously mapped to pheS (Hennecke & B&k, 1975); therefore, it was decided to clone and sequence the mutant pheS genes. To functionally test whether the identified mutation site carries determinants important for Phe binding, a series of sit,e-directed mutagenesis experiments were carried out. The results obtained led to a model, which assumes that residue 294 of the PheRS a subunit is part of the Phe binding site. Furthermore, the

Synthetase Specijkity

101

model could explain the mechanism for discrimination against p-F-Phe in the wild-type enzyme and led to the construction of a PheRS mutant with relaxed specificity towards selected Phe analogues. 2. Materials (a) Bacterial

and Methods phages and DNA

strains,

The bacterial strains, vectors and plasmids used in this work are listed in Tables 1 and 2. A phage Plkr lysate from strain JC10289/pKY102 (Ihara el al., 1985) was obtained from A. Birkmann (University of Munich, Germany). VCS-Ml3 helper phage for single-strand DNA isolation was from Stratagene (San Diego, CA. U.S.A.). Oligonucleotides used for mutagenesis and conventional (radioactive) sequencing were synthesized on an Applied Biosystems DNA synthesizer model 380B. pheS-specific dye-primers for automated DNA sequencing were kindly provided by G. Zon (Applied Biosystems. Foster City, CA, U.S.A.). Dye-primer -36M13 was purchased from the same company. (b) Substrates

and annlogu,es

p-Fluoro-L-phenylalanine and prot’einogenic amino acids were from Fluka Chemie AG (Buchs. Switzerland), and p-fluoro-n,r,-phenylalanine (p-F-Phe) from Sigma Chemicals (St Louis, MO, U.S.A.). o- and m-flUOrO-D,Lphenylalanines, 3-flUOrO-D,L-tyI’OSiUe and all 6 di-fluoro substituted n,L-phenylalanines were kindly provided by P. Babczinski (Bayer AG. Leverkusen, Germany). pDeutero-. p-chloro-. m-chloro- and p-bromo- derivatives of L-phenylalaninr and P-thien-2-vl-L-alanine were synthesized by Sergio Cantoreggi f&m I). Seebach’s group (Laboratorium fiir Organ&he Chemie. ETH Ziirich; Seebach et al.. 1989). I.-Phenyl-[ l-‘4C]alaninr (20 mCi/mmol) and p-fluoro-o.L-[l’-‘4C]phenylalanine (4 mCi/mmol) were from Amersham (I’.K.) and CIS (Gif-sur-Yvette. France), respectively. (c) Microbiological

techniques

E. coli strains were usually grown in LB-medium (Miller, 1972). The antibiotics used were ampicillin at 150 pg/ml, tetracyclin at 15 pg/ml or streptomycin at 100 pg/ml. Construction of the rec& strain KA2 was accomplished by generalized transduction according to the protocol given by Miller (1972). A Plkc lysate from the recA- deletion strain JC10289/pKY102 was used, which allowed cotransduction of the recA- marker and a tetracyclin resistance gene (Ihara et al., 1985).

(d) In vivo plate tests In vivo plate tests for thermoresistance

(i.e. complementation of strain KA2) were performed by streaking out single colonies on LB-agar plates containing appropriate antibiotics. Growth was recorded after 24 and 48 h of incubation at 30°C or 40°C. When full expression of plasmid genes from the lac promoter was required, IPTG was added to @5 mM final concentration. Plate tests for the analysis of Phe-analogue resistance were done on minimal medium plates containing 100 mm-KH2P04, 12 mM-(NH,),SO,, 50 mM-Na,HPO,, 1 mM-MgSO,, @l mM-CaCl, and @4% (w/v) D-&COW in 1.5% (w/v) agar. The medium was supplemented with thiamineHCl (5 pg/ml) and, when required, with amino acids (50 &ml), other vitamins (5 pg/ml), ampicillin

102


Table 1 E. coli strains and plasmids used in this work

E. wli strains

Genotype

AB1360-12

K12, F-, pheSl2, t&l, argE3, his-l, proA2, aroD6, lacH1, galK.2, m&l, xyk-5, k-29, supE441, Iz-, str KlO, Hfr(Cavalli), pheSl3, Tel-l, kmA22, thi T2= K12, F-, pheSI2, recA, Ihi-1, leu-6, proA2, hadS20 (rim,), lacY1, galK2, ara-14, m&l, xyl-5, wpE44, rpeL20 (&r-20), endA, IKlO, Hfr(Cavalli), ph.&G, rel-1, tonA22, thi, T2R

KIO-F6 RR28

NP37 KA2

KlO, Hfr(Cavalli), pheS5, rel-12, taA22, thi, T2” A(arlR-recA)306 : : TnlO K12, F-, recAl3, thi-I, leu-6, proA2, hadS20 (rim&), lacYI, galK2, ara-14, mtl-1, xyl-5, supE44, Tp8L20 (&r-20), endA, AK12, recA-, endAl, gyrA96, thi, hsdRl7, supE44, relA1, A(luc-proAB), F’[traD36, proAB+, 1aeP lacZAMIS] K12, hsdA5, thi, supE, A(luc-proAB), F’[traD36, proAB+, lacP, lacZAMl5]

HBlOl

JM109

TGl

Vectors

Relevant

pACYCl84 pBluescript KS ( + ) ( =pBLS) puc19

pKSC-X

pKSBl-X

pKSB2-X

Reference or origin

p-F-PheR, StrR

Hennecke & B&k

(1975)

p-F-PheR

Hennecke & B&k

(1975)

p-F-PheR, RecA-, HsdR-M-, Strs

Hennecke et al. (1982)

PheRS”

Eidlic & Neidhardt (1965); Back & Neidhardt (1967); Comer & B&k (1976) This work: Pl transductant of NP37

PheRS”, RecA-,

Tet”

St2

Boyer & Roulland-Dussoix Sambrook et al. (1989)

RecA-, HsdR-M+, for cloning

host Yanisch-Perron

HsdR-M-, host for single-strand DNA isolation

Relevant

(1969);

et al. (1985)

Sambrook et al (1989); purchased from Amersham (U.K.)

properties

Reference or origin

CamR, Tet’; a derivative of plasmid P15A AmpR; a derivative of pUCl9 carrying a polylinker and the intergenic region of phage fl Amp”; a derivative of pBR322 with a polylinker

Plasmids pHE3 pHW0

Relevant phenotype and application

Chang & Cohen (1978) Short et al. (1988); purchased from Stratagene (San Diego, CA, U.S.A.) Yanisch-Perron et al. (1985); purchased from Pharmacia (Uppsala, Sweden)

properties

Vector

Reference or origin

CamR, pheS (wild-type) AmpR, TetR; containing E. coli thrS, injC, rpml, rplT, pheS, pheT on 1025 kb EcoRI-Hind111 fragment AmpR; containing thrS, infc, rpml, rplT, pheS*, pheT on 1025 kb EcoRI-Hind111 fragment from E. wli pheS mutant strains

pACYC184 pBR322

Hennecke et al. (1982) Elhardt et al. (1982); [=pBl Plumbridge et al. (1980)]

puc19

AmpR; containing pheS* on 1181 bp Seal-Hind11 fragment; orientation: pheS* in phase with lac promoter As pKSBl-X, but pheS* in opposite orientation (not expressed from Zac promoter)

pBluescript

KS (+ )

pBluescript

KS (+ )

This work (Fig. 1; -X stands for pheS genes from strains KlO-F6 (-K), AB1360-12 (-A) and wild-type (-W, ex PHWO) This work (Figs I(b) & 3); -X stands for strain abbreviations (see pKSC-X) or mutation type (Table 2) This work (Figs l(b) & 3); -X stands for abbreviations as described for pKSBl-X

in

An asterisk (*) indicates the presence of different pheS alleles. Gene symbols are according to Bachmann (1990). Amp, ampicillin; Cam, chloramphenicol; Str, streptomycin; Tet, tetracyclin

(150 pg/ml) and IPTG (@25 mM). The Phe analogues were present at a concentration of O-55 mM (of the L-enantiomer). Growth at 28°C or 37°C was monitored over a period of 4 days. All tests were performed in duplicate and repeated at least once. (e) Recombinant

DNA

techniques

and DNA modifying Restriction endonucleaaes enzymes were purchased from Boehringer (Mannheim, Germany) or Pharmacia (Uppsala, Sweden) and used by the supplier. [a-32P]dATP as recommended (3000 Ci/mmol) was from Amersham (U.K.). Cloning, transformation, plasmid isolation and nick-translation were performed using standard procedures (Maniatis et al., 1982; Sambrook et al., 1989). Single-strand DNA of Bluescript

plasmids

(pKSB1/2)

in strain

TGI

was isolated

using a scale-up of the method described by Stratagene (San Diego, CA, U.S.A.). For Southern-blot analyses (Southern, 1975), the protocols described by Maniatis et al. (1982) were followed. Oligonucleotides were purified on a 12% (w/v) polyacrylamide gel as described (Sambrook et al., 1989). Oligonucleotide-directed mutagenesis was carried out according to the in vitro method of Nakamaye & Eckstein (1986) using the kit and detailed protocol from Amersham (U.K.). The mutagenic oligonucleotides (listed in Table 2) were synthesized with mixed base addition at suitable positions to create up to 4 different mutations in 1 redundant synthesis run. (f) Cloning Chromosomal

strains

DNA

using standard

of mutunt

pheS genes

wa.s isolated

procedures

from

mutant

E. c&i

(Schleif & Wensink,

Altered Phenylalanyl-tRNA

Synthetase Specijicity

103

Table 2 Oligonucleotides and corresponding mutations Oligonucleotide (-mixture) Name

Sequencet

Size

WiM-type MA-ST

20mer 5’-CTCTGGlTE(T/A)CCTTCGGGA-3

20mer 5’-CTCTGGlTKTGCllCGGGA-3

MFl-GV 20mer 5’-TACTCTGGTG(G/T)CGCCllCGG-3

MFI-YW 20mer S-TACTCTGG~(A/G)(C/G)GCC~CGG-3

MF2-GV 20mer 5’-GGlTEGCCG(G/l)CGGGATGGG-3

MF2-YW 20mer 5’-GGlTfCGCCT(A/G)(C/G)GGGATGGG-3

aa-Sequence? 292 to 296f

Plasmid pKSH1/2-

...GlyPheAlaPheGly... -W

[877]-TTC GCC l-K-[SSS]

MA-GV 20mer 5’-CTCTGGTTTCG(G/l)CllCGGGA-3

MA-C

Mutated codonst (mutation)1

GCC+TCC (680)

...GlyPheSerPheGly... -M4S

GCC+ACC (880)

...GlyPhel’hrPheGly... -M4T

GCC+GGC (881)

...GlyPheGlyPheGly... -M4G

GCC+GTC (881)

...GlyPheValPheGly... -M4V

GCC-BTGC (880/l)

...GlyPheCysPheGly... -M4C

TTWGGC

(877/a)

...GlyGlyAlaPheGly... -M3G

llC+GTC

(877)

...GlyValAlaPheGly... -M3V

lTC+TAC

(878)

...GlyTyrAlaPheGly... -M3Y

llC+TGG

(878/g)

...GlyTrpAlaPheGly... -M3W

llC+TGC

(878)

...GlyCysAlaPheGly... -M3C

TTWGGC

(88314)

...GlyPheAlaGlyGly... -M5G

lTC+GTC

(883)

...GlyPheAlaValGly... -M5V

llC+TAC

(884)

...GlyPheAlaTyrGly... -M5Y

l-fC+TGG

(884/5)

...GlyPheAlaTrpGly... -M5w

llC+TGC

(884)

...GlyPheAlaCysGly... -M5C

l-K+TAG

(88415)

...GlyPheAla

-M5

lTC+GGC, llC+GGC (877/8,883/4) ...GlyGiyAlaGlyGly... -M3G5G 2MF-GV 26mer 5’-TACTCTGGTG(T/G)CGCCG(T/G)CGGGATGGG-3’

2MF-W 26mer 5’-TACTCTGGllGGGCCTGGGGGATGGG-3

ITWGGC, TTC+GTC (87718,883)

...GlyGiyAlaValGly... -M3G5V

l-K+GTC,

llC+GGC (877,88314)

...GlyValAlaGiyGly... -M3V5G

llC+GTC,

l-K+GTC

...GlyValAlaValGly... -M3V5V

(877,883)

lTC+TGG, TTC+TGG (878/9,884/5) ...GlyTrpAlaTrpGly... -M3W5W

iBold fare nucleotides or amino acids indicate the point mutations introduced. fNumbering of nucleotides and amino acids is according to Fayat et al. (1983).

1981) and then digested with EcoRI and HindIII. The presence of the 1925 kb EcoRI-Hind111 fragment carrying pheS (see Fig. 1) was verified by Southern blot hybridization. The radiolabelled probe was nick-translated plasmid pHE3; the vector alone (pACYC184) served as control. DNA fragments from the 10 kb region were cut out from a preparative agarose gel and ligated to a pUC19 vector digested with EcoRI and HindIII. After transformation of the thermosensitive E. coli strain NP37 @he&““), selection at high temperature (40%) on ampicillin plates allowed only growth of cells containing pheS on plasmids (plasmids pKSC-X; see Table 1). (g) DNA

sequence analysis

The pheS DNA was completely sequenced on both strands using the dideoxy chain termination method of

Sanger et aE. (1977). Oligonucleotide primers (6 per strand; sequences not shown) were synthesized according to the available pheS wild-type sequence. They covered sites separated by about 250 bp from each other and permitted the overlapping sequencing of the entire mutant pheS genes in parallel to that of the wild-type. The sequencing reactions were carried out with singlestrand DNA using the Klenow fragment of DNA polymerase I (Pharmacia, Uppsala, Sweden) or SequenaseTM (Tabor & Richardson, 1987; United States Biochemical Corp., Cleveland, OH, U.S.A.). The pheS coding strand was sequenced with [a-32P]dATP using the conventional methods described by Sambrook et aE. (1989). For the opposite strand, phefkpecific 5’-end-labelled dye-primers were used that allowed automatized sequencing (Smith et al., 1986; Connell et al., 1987) with the DNA Sequencer model 370A, following protocols provided by the manu-

104


facturer (Applied Biosystems, Foster City, CA, U.S.A.). Computer-assisted sequence alignments were done using programs of the University of Wisconsin Genetics Computer Group (UWGCG; sequence analysis software package release 6.2; Madison, WI, U.S.A.). (h) Preparation and analysis of protein Sl40 extracts A bacterial culture was grown at 30°C by vigorous shaking to an absorbance (&cc) of 2.0 in 300 ml rich medium (1 o/o (w/v) bactotryptone, O-5% (w/v) yeast extract, @2% D-glucose and appropriate antibiotics; @5 rnM-IPTG was present if plasmid-encoded pheS had to be expressed). Then, the 5140 protein extract was prepared essentially as described by Griill et al. (1979). After ultracentrifugation, the protein concentration was determined by the Folin phenol procedure (Lowry et al., 1951) with bovine serum albumin (Sigma Chemicals, St Louis, MO, U.S.A.) and a reference extract as standards. The assays were repeated several times and the calculated values (Peterson, 1983) typically had a standard deviation of 5% or less. The content of PheRS fl and a subunits was monitored by Western-blot analysis (Burnette, 1981) after separation of protein samples on a 10% polyacrylamide gel containing @l% (w/v) SDS (Laemmli, 1970). Proteins were either stained with Coomassie blue or transferred to a nitrocellulose membrane (Hybond-C; Amersham, U.K.) employing the Mini Protean II Dual Slab Cell and the corresponding transfer cell from Bio-Rad (Richmond, CA, U.S.A.). As primary antibodies, rabbit antisera (y-immunoglobulin fraction) raised against wild-type PheRS or isolated a subunits (Hennecke et al., 1977b) were used. The immunological staining of the membrane was done essentially as described in the Immun-Blot Assay protocol (1988) from Bio-Rad using goat anti-rabbit IgG/alkaline phosphatase conjugate as secondary antibody. As standards, a pre-stained protein size marker (Bio-Red) and purified E. coli PheRS protein (kindly provided by H. Sternbach, Max-Planck-Institut, GGttingen, Germany) were used. Stained gels and membranes were scanned in a Molecular Dynamics Computing Densitometer model 300A (Sunnyvale, CA. U.S.A.). (i) Determination

of PheRS activity in vitro

(i) Standard assay The PheRS assay was based on the methods given by Kosakowski & B&k (1970) and Comer & B&k (1976). Enzymatic activity was measured by attachment of L-[14C]Phe to tRNA. The reaction mixture contained (in a total volume of 250 ~1) 100 mm-Tris. HCl (pH 7.5), 10 mM-KCl, 10 m&i-M&l,, 2 mM-reduced glutathione, 2 mM-adenosine triphosphate, 2.4 mg E. coli tRNA/ml (from strain MRE600; Boehringer, Mannheim, Germany) and 20 pr+L-[14C]Phe (20 mCi/mmol). After addition of 5140 protein extract (usually 4 to 8 pg), the mixtures were incubated at 28°C for various time intervals. The reaction was then stopped by chilling and precipitation of tRNA with 10% (w/v) trichloroacetic acid containing 1 mg n,L-Phe/ml and further processed as detailed by Kosakowski $ Bijck (1970). For incorporation of p-F-Phe, 40 pM-D,L-p-F-[‘4C]Phe (4 mCi/mmol) was used instead of L-[14C]Phe. Initial velocities were determined graphically from at least 5 independent data points taken from 0 to 10 min; background slopes (without enzyme) were subtracted. All test series were repeated at leaat once, the deviations of initial velocities usually being less than 5%.

(ii) Analysis of mutant PheRS activities Initial velocities of aminoacylation (expressed in nmol Phe attached to tRNA per min and mg protein) in 5140 extracts from KA2 cells harbouring mutant pheS plasmids were determined at 28”C, and also at 40°C and 45°C (with or without preincubation of extracts at 1 mg protein/ml concentration for 20 min at 45°C). Values of certain thermosensitive mutants (marked in the Figs) could not be determined accurately (within 5 to 10% error; indicated in percent of the normal wild-type value) at high temperatures. For rough approximations of apparent KM values, Phe concentrations of 4 PM, 20 PM and 80 pM were used. Each data point was measured twice (incubation for 5 or 10 min at 28°C) and individual backgrounds were subtracted for each Phe concentration. The results were analysed by Lineweaver-Burk plots (Fersht, 1985a); the error was estimated to be 25%. From these KM data, approximate apparent k,,, values were deduced from the Michaelis-Menten equation (Fersht, 1985a) using the initial velocities at 28°C and the total enzyme concentration as estimated by comparison (of the /l subunit band) to known PheRS standards on Western blots. The effect of a change in substrate specificity of mutant PheRSs was assessed by performing the standard assay (5 or 10 min incubation at 28°C) in the presence of a 50-fold excess (corresponding to 1 mM of the L-enantiomer) of one or more potentially competing substrates. Differential p-deutero-L-phenylalanine effects of competition compared to the natural substrate were analysed by titrating both substances over a concentration range of 0.5 pM to 1 mM. All competition assays were carried out at least twice. The value of remaining [ 14C]Phe incorporation was compared to the activity in the absence of a potentially competing substrate ( = 100%). Deviations among independent measurements were generally less than 10 to lS?/, of the value for non-competed incorporation.

3. Results (a) Cloning

and sequencing of mutant pheS genes

To determine the mutations responsible for the p-F-Phe ex c 1u d ing mutant PheRSs, the pheS gene EcoRI-Hind111 fragments, regions (1@25 kb Fig. l(a); Fayat et al., 1983) were cloned from chromosomal DNA isolated from the two spontaneously obtained p-F-PheR strains AB1360-12 and KlO-F6. The cloning was achieved by complementation of the pheS mutant strain NP37 (thermosensitive PheRS) as described in Materials and Methods. The resulting plasmids pKSC-A and pKSC-K contained the fragments from strains AB1360-12 and KlO-F6, respectively. pKSC-W was constructed by cloning the corresponding wild-type EcoRI-Hind111 fragment from plasmid pHW0 (Table 1) into the vector pUC19 in analogy to the procedure for the mutant genes. As shown in Figure l(b), the pheS-containing SmaI-Hind11 fragments from the pKSC plasmids were subcloned in both orientations into the sequencing and expression vector pBluescript KS (+) to obtain the plasmid series pKSBl-X and pKSB2-X (Table 1). Both mutant pheS genes were sequenced parallel to the wild-type pheS gene on


Synthetase Specificity

105

Table 3 PheRS activity of pheS mutants tested in vivo and in vitro A In viva growth tests? Rich medium (LB) Strains carrying chromosomal pheS only

30°C

40°C

HI%101 (wild-type) ABl36@12 (p-F-PheR) KlO-F6 (p-F-PheR) RR28 (p-F-PheR) KA2 (PheRS”)

++ ++ ++ ++ ++

++ k$ ++ ++ 0

MMS, 28°C

PheRS activityg

-p-F-Phe

+p-F-Phe

[“‘CjPhe

p-F-[ ‘%]Phe

+I ++ ++ ++ n.t.11

0 ++$ + + n.t.11

308 2.54 2.48 n.t.l( 1010

1.53 1@03 10.09 n.t.(l I 0.08

I3 In wiwo growth tests? In strain KA2 (PheRS’%) LB, Amp, IPTG Plasmids carrying pheS (expected phenotype) pULS (vector) pKSBl-W (wild-type) pKSB 1-A (p-F-Phes) pKSBl-M4S (p-F-Phes)

30°C

40°C

++ ++ ++ ++

0 ++ ++ ++

In strain RR28 (p-F-Phe”, recessive) MM$, Amp, IPTG; 37°C

PheRS activity in plasmid-bearing KA2 strain5

-p-F-Phe

[14C]Phe

++ ++ ++ ++

+ p-F-Phe + + +

+ > + > - >O. Amp, ampicillin. $ The minimal medium (MM) of each strain w&5 supplemented according to its requirements (Table 1). Individual strains may behave differently in viwo due to their dissimilar genetic backgrounds. §Activity is given in nmol Phe or p-F-Phe attached to tRNA per min and mg protein in 8140 extracts at 28°C‘ 11n.t.. not tested.

both strands as described in Materials and Methods, and the deviations from the published wild-type pheS sequence (Fayat et al., 1983) were determined. At codon position 74, an arginine triplet (CGT) was found instead of the one for alanine (GCT) published by Fayat et al. (1983). The fact that this deviation was present in all three independent genes isolated from different strains (including the wildtype) suggests t’hat the former alanine codon was due to an error in the previous sequence determination (C/G switch at nucleotide position8 2201221). The Arg tits better into the sequence alignment with the homologous PheRS small subunits from yeast cytoplasm, yeast mitochondria and Bacillus subtilis, in which either Arg or another basic amino acid (Lys) is present at that position (Brakhage et al., 1990). Another sequence alteration was found in both pheS genes from the two independently isolated p-F-Phe-resistant strains AB1360-12 and KlO-F6, but not in wild-type pheS. It consisted of a G to T transversion at nucleotide 880 in each mutant pheS gene leading to an alanine-to-serine exchange at amino acid position 294. (b) An Ala-to-Ser exchange at position 294 is responsible for the p-F-PheR phenotype in vivo and in vitro The Ala-to-Ser mutation only deviation between p-F-Phe-resistant PheRS

(termed S294) was the the wild-type and a a subunit. To prove

unambiguously that S294 is responsible for the p-F-PheR phenotype, a series of in vivo and in vitro test8 were carried out to compare the parental strain8 with strains carrying the cloned pheS genes on plasmids. In addition, the 5294 mutation was created again in a wild-type gene by oligonucleotide-directed mutagenesis (see below) yielding plasmid pKSBl-M4S (Table 2). To assay the individual pheS gene products, the pKSBl-X plasmids (which allowed relevant expression of the pheS genes from the lac promoter, Fig. l(b)) were introduced into strain KA2, a RecA- derivative of NP37 constructed by generalized Pl transduction. The pheS genes were fully expressed by addition of IPTG, and the PheRS phenotype was assayed by in vivo growth tests (on agar plates) as well a8 in vitro (protein extraction and PheRS assay) as described in Material8 and Methods. The results are compiled in Table 3, together with those obtained with the parental strains. In addition, Table 3 contains data from the pKSB1 plasmids in the p-F-PheR strain RR28. In this background, the presence of a wild-type pheS allele causes the p-F-PheR strain to become sensitive to the Phe analogue. This dominance of the wildtype gene is due to deleterious p-F-Phe incorporation into cellular protein (Fangman & Neidhardt, 1964; Hennecke et al., 1982, and references therein). Growth failure of transformed RR28 on p-F-Phecontaining plates, therefore, is an indication for a wild-type pheS gene present on t’he plasmid.

106

P. Kast and H. Hennecke s294 1

‘Phe binding region’ I

Escherichia wli

vvv 285.,.1-l

VlY

S G F A F G M G M E R L T M L R Y1...307

Yeast cytoplasm Motif 3 consensus PheRS consensus Figure 2. Alignment of PheRS small subunit sequences corresponding to the pheS region around the position of mutation 5294. The pheS gene products from E. coli (Fayat et al., 1983), B. subtilis (Brakhage et al., 1990), yeast mitochondria (Koerner et al., 1987) and yeast cytoplasm (Sanni et al., 1988) were aligned using the UWGCG program CLUSTAL (Higgins & Sharp, 1988). Furthermore, we included a corresponding protein sequence derived from a partial pheS sequence of S. typhimurium (B. Keller & P. Kast, unpublished results). Numbers indicate the first and the last residues shown here for the respective sequences. The l-letter code for amino acids is used. Identical residues are boxed; thin lines connect related amino acids with a pairwise value of at least 97 according to the Dayhoff mutation data matrix (Dayhoff et al., 1983) resealed as indicated in the UWGCG sequence analysis software package, release 62. The PheRS consensus features around position 294 of the E. coli sequence are indicated. Residues of E. coli PheRS mutagenized in this work are specified by arrowheads. In addition, the motif 3 consensus sequence of class II tRNA synthetases is aligned (adapted from Eriani et al., 1990a). Lower case letters denote predominantly occurring residues; the arginine is completely conserved; Cpstands for hydrophobic amino acids if they are present in more than 70% of the sequences examined by Eriani et aE. (1990a).

The results in Table 3 show that strains containing plasmid pKSBl-A behaved like the p-F-PheR parental strains AB1360-12 and KlO-F6 on Phe-analogue plates (i.e. growth) and in the PheRS assay (i.e. no significant analogue incorporation).

Identical

results

were obtained

with

plasmid

pKSBl-M4S, which proved that the mutation S294 was solely responsible for the p-F-PheR phenotype. The plasmid pKSBl-W, as expected, conferred p-F-Phe sensitivity to RR28, and the PheRS values obtained in vitro agreed with those of HBlOl, the and wild-type reference strain. Both wild-type mutant pheS genes allowed complementation of the PheRS’” strain KA2. It is worth mentioning here that the host strain KA2 showed virtually no background activity in vitro at 28°C (Table 3).

(c) Strategies for site-directed mutagenesis of residues around position 294 (i) A working model for the phenylalanine binding site in PheRS As shown in the sequence alignment in Figure 2, the mutation S294 mapped to the C terminus of the E. coli PheRS u subunit precisely within motif 3, a conserved region in class II tRNA synthetases (Eriani et al., 199Oa). A closer inspection of motif 3

around position 294 showed a particularly high degree of conservation within the known PheRS small subunit sequences from different organisms. Figure 2 contains, in addition to the relevant E. coli sequence, the corresponding regions of the previously sequenced PheRS small subunits from yeast cytoplasm and mitochondria, and B. subtilis. Furthermore, we included the equivalent sequence from Salmonella typhimurium, a close relative of E. coli; its pheS gene was cloned recently in our laboratory by a strategy analogous to that described in this work for cloning the mutant pheS genes (B. Keller and P. Kast, unpublished results). The presence of the p-F-PheR mutation within a PheRS-specific conserved region prompted us to localize there at least part of the Phe-binding site. Furthermore, assuming that the residue at position 294 contacts the substrate phenylalanine, it seemed possible that the two amino acids immediately flanking position 294 might also participate in substrate binding. As a working hypothesis we considered a model in which the aromatic rings of residues 293 and 295 (which are both phenylalanine residues; Fig. 2) could interact with the aromatic side-chain of the substrate phenylalanine. The model implied that residues 293 to 295 form part of a hydrophobic pocket for phenylalanine, whereas

the para-position

of the substrate’s

aro-

Altered Phenylalanyl-tRNA contact residue matic ring 294. would Perpendicularly interacting aromatic residues may contribute substantially to the stabilization of a protein structure (Burley & Petsko, 1985, 1986; Hunter et al., 1991; Serrano et al., 1991). Apart from this, aromatic amino acids are often found to participate in the formation of a hydrophobic pocket designed to bind an aromatic substrate (Hangauer et al., 1984). (ii) Strategies for amino acid replacements To test our working model of the phenylalanine binding site, the amino acids in positions 293, 294 and 295 of the PheRS a subunit were exchanged against selected other amino acid residues via sitedirected mutagenesis. The choice of these amino acid replacements was also influenced by the similarity in that region within PheRS sequences from different origins. As shown in Figure 2, the consensus features of residues at positions 293, 294 and 295. are large hydrophobic, small and aromatic, respectively. Consequently, the amino acid replacements in E. coli PheRS were designed to systematically test and alter this conserved pattern. The strategy for mutagenesis and subsequent analysis of mutant a subunits is presented in Figure 3. Mutagenic oligonucleotides (or oligonucleotide mixtures), the resulting mutant pheS gene plasmids and the exchanged amino acid residues are listed in Table 2. (iii) Precautions made in the subsequent analysis of mutants To measure the activity of mutated PheRS in vivo and in vitro, two peculiarities of the protein were taken into account: (1) Since PheRS is an essential enzyme, mutant plasmids had to be transformed into a conditional mutant. The thermosensitive E. coli strain KA2 was chosen as it showed no PheRS activity in vitro even at 28°C (Table 3). (2) To avoid an interference of the chromosomally encoded thermosensitive a subunit of strain KA2 in the assays, the gene dosage for the proteins to be tested was drastically increased by introducing the pheS genes on a multicopy plasmid; in addition, the genes were expressed from an induced lac promoter (Fig. 3). Nevertheless, the plasmidial pheS expression did not affect the amount of PheRS holoenzymes produced because both a and fl subunits were needed to form an active enzyme (Hennecke & B&k, 1975) and the chromosomally encoded fl subunit was limiting. This system allowed a direct comparison of the specific enzyme activities obtained in the aminoacylation tests with the various mutant S140 protein extracts. The validity of the concept was confirmed experimentally by the high reproducibility of specific activity values, irrespective of strain type and plasmidial or chromosomal location of the assayed pheS genes (Table 3). It was further confirmed by Western blot analyses (data not shown), which showed that the concentration of

Synthetaee Specificity

107

(activity-limiting) fl subunit was essentially the same in all S140 extracts. By correlating the scanned intensities of B bands from 5140 extracts with those of the PheRS standards, a typical concentration of @029 nmol PheRS per mg S140 extract was obtained. Based on this and on the determinations of apparent KM values (see below), the calculated apparent k,,, for aminoacylation by wild-type PheRS was about 150 per minute (at 28°C). This value is in good agreement with the previously determined k,,, for aminoacylation by purified E. coli PheRS of 228 per minute (at 25”C, analogous assay conditions; Holler, 1980). Unexpectedly, the a subunits of PheRS were not always present at a high level when compared to controls without plasmid pheS genes. The amount of a subunit varied between 1.1 and 8.3 times the value of the host strain KA2. This phenomenon was observed with three different antisera raised against the a subunit or the PheRS holoenzyme (data not shown) and was supposed to be due to degradation of excess free a subunits (see Discussion). The relative a subunit levels (not shown) did neither correlate with the PheRS activity/thermosensitivity in the S140 extracts nor with the mutation type. The presence of significant amounts of hybrid enzymes (containing one chromosomally and one plasmid-encoded a subunit; Hennecke, 1976) could be ruled out by thermoinactivation tests (data not shown) as described by Hennecke (1976). Additional substrate competition experiments carried out at 45°C (after preincubation at that temperature to inactivate hybrid enzymes; Hennecke, 1976) with several of the partially thermolabile mutants (see below) gave results (not shown) identical to those obtained at 28°C.

(d) Replacing phenylalanine

at position 293

To test the hypothesis of an aromatic interation between the presumed amino acid binding site of PheRS and the side-chain of the phenylalanine substrate, Phe293 was exchanged against Gly, Cys, Val, Tyr or Trp. All mutants were active in complementing the thermosensitive strain KA2 (Fig. 4(a)), and in vitro activity at 28°C showed a maximal reduction of 30% for the Gly293 and Trp293 variants relative to the wild-type Phe293 (Fig. 4(b)). With a wild-type pheS gene in KA2, we obtained a KM value for Phe of 7 PM, which is in excellent agreement with the value of 6.6 ,UM obtained previously under similar conditions for a wild-type strain (Griill et al., 1979). This result was further confirmed with our PheRS wild-type strain HBlOl (data not shown). Figure 4(c) shows that KM values for Phe were not significantly affected by the Together with the other data in mutations. Figure 4, this suggests that Phe293 (or an aromatic residue at that position) is not important for phenylalanine binding or PheRS activity. At higher temperature, the reduction of in vitro enzyme activities amounted to 58% (Gly293), as

1 Western-blots]

A

140 extracts

i=

I

Aminoacylation assays Avitro phenotype

Figure 3. Experimental strategy for oligonucleotide-directed mutagenesis of pheS genes and subsequent analysis of mutant PheRSs. Single-strand DNA of pKSB2-W carrying the wild-type pheS gene was mutagenized using mutagenic oligonucleotides listed in Table 2 (see Materials and Methods). The resulting plasmids (pKSB&X; in which X stands for the different mutants in Table 2) were isolated as single strands from strain TGl and sequenced to screen for mutations in the region around codon 294. From plasmids containing the desired mutations, a 174 bp A&II-B&B1 fragment (marked by a flash arrow) was excised and used to replace the corresponding fragment in the wild-type pheS plasmid pKSBl-W. Strain RR28 was transformed with the ligation mixture. The resulting constructs (pKSBl-X) were suited to express the mutant pheS genes from the Zucpromoter (P,,,C)of pBluescript after addition of the inducer IPTG. Plasmids isolated from strain RR28 were transformed into strain TGl and subsequently sequenced. In parallel, the pKSBI-X plasmids were modified by passage through the EcoK modification-proficient strain JM109 and finally introduced into the tester strain KA2, which carries a pheS gene for a thermosensitive PheRS. The in. viva phenotypes of RR28 and KA2 cells transformed with pKSBl-X were analysed on agar plates. Western blots and in vitro assays of mutant PheRS activities were carried out with S140 protein extracts of KA2 transformed with pKSBl-X. An arrow in front of the mutagenized fragment indicates the position of the sequencing primers. bla, gene for /?-lactamase conferring resistance to ampicillin; ssDNA, single-stranded DNA. Only the relevant genotypes/phenotypes for the strains used are indicated; a complete listing is provided in Table 1.


109


(a)

In who activity: complementation of KA2 at 40°C

(b)

Initial velocities of aminoacylation at different temperatures

Aminoacylation activity (nmol Phe / min mg)

pBLS Gly

(C)

Cys

Val

Phe Tyr WI

Trp

Approximate kinetic parameters at 28°C

Apparent bt (1/min)

KM for Phe (PM)

160

10 9

140

0 120

; n kcat 5 H&l

100

00 00

4 3

40 2 20

1

0

0

Gly

Cys

Val

Phe Tyr

Trp

Figure 4. Phenotypic effects of amino acid exchanges at position 293 of the PheRS CIsubunit. The amino acid variants are arranged in the order of increasing surface area of the residue at the corresponding mutated position (Rose et al., 1985). wt. wild-type; pBLS, negative control (vector only). (a) The ability of plasmid-borne pheS variants to complement the thermosensitive strain KA2 was scored from good ( + +), normal (+), reduced (+), marginal ( - ) growth to no growth at all (0). (b) Initial velocities of Phe aminoacylation by S140 extracts from KA2 cells carrying mutant pheS plasmids. The standard assay was carried out at the temperatures indicated. The values 2g”C, p.i. were obtained with 5140 extracts preincubated at 45°C for 20 min and then cooled to 0°C prior to assaying at 28°C. The errors are estimated to be 5 to 10% relative to the wild-type values. (c) Apparent k,,, and K, values at 28°C calculated as described in Materials and Methods. For determination of k,,, the average of initial velocities at 28°C (with and without preincubation) from (b) was taken. The values represent only rough approximations (estimated errors 2506).

110


compared to the wild-type level (Fig. 4(b)). With the exception of Cys293, there was a tendency to favour a residue with a surface area comparable to Phe (especially at high temperature). The slightly thermosensitive behaviour of some mutants was not due to irreversible thermal denaturation, since preincubation of S140 extracts at 45°C (and subsequent cooling to 0°C) did not alter activity at 28°C (Fig. 4(b)). This feature was also apparent with all mutants analysed below. (e) Replacing phenylalanine

at position 295

It was also tested as to whether the conserved aromatic residue at position 295 (again a phenylalanine; Fig. 2) was essential for PheRS activity. The data in Figure 5 demonstrate that the effect of point mutations at this position was more pronounced than with Phe293. Mutant Gly295 could not complement strain KA2 (Fig. 5(a)) and showed low PheRS activity in vitro (19%, 3% and 0% of wild-type values at 28 “C, 40°C and 45°C respectively; Fig. 5(b)). Mutations to Cys295 and Va1295 allowed the formation of complementing a subunits and exhibited nearly wild-type activity at 28°C (Fig. 5(a) and (b)). Exchanging Phe295 against Trp represents a fully tolerated alternative for E. coli PheRS. The aromatic exchange to Tyr295 yielded wild-type activity in viva and in vitro at 28”C, but showed, like Cys295 and Va1295 variants, a strong thermosensitive behaviour at 40°C and 45°C (Fig. 5(a) and (b)). The Khl values were not changed (Fig. 5(c)). Insertion of a nonsense codon at position 295 (mutant M5) abolished PheRS activity completely (Fig. 5(a) and (b)). The absence of crossreacting material corresponding to this truncated a subunit in the 5140 extract (not shown) suggested that this defective protein was rapidly degraded. (f) Replacing both phenylalanine residues at positions 293 and 295 To rule out the possibility that one of the two aromatic residues alone was sufficient for binding of the phenylalanine substrate, both Phe293 and Phe295 were exchanged simultaneously. The data in Figure 6 confirmed that a glycine at position 295 was not tolerated as already seen in the previous section (see Fig. 5). Activity values were even more reduced in vitro, when Phe293 was additionally replaced by Gly or Val (Fig. 6(b)). The fact that the double mutants Val293/Val295 and Gly293/Va1295 could complement KA2 (Fig. 6(a)) and were active at 28°C (albeit with reduced rate) proved that aromatic residues at positions 293 and 295 are not at all essential for PheRS activity and, in particular, substrate binding. This was further supported by the absence of significant alterations in the apparent K, values for the double mutants, perhaps with the slight exception of the Trp293/Trp295 variant (Fig. 6(c)). In addition to the strongly reduced activities at

40°C and 45°C of some mutants (Figs 5(b) and 6(b)), a pronounced decline of aminoacylation with time was observed in certain cases (data not shown). This effect appeared exclusively in assays carried out at high temperatures and was seen for all mutants having Gly or Val at position 295 as well as for the Trp293/Trp295 double mutant. (g) Do replacements at positions 293 and 295 change the substrate specificity? An independent means to screen for phenylalanine binding site mutants is the examination of the substrate (or substrate analogue) spectrum of the different PheRS variants. The plasmids carrying the mutant pheS genes were analysed in E. coli strain RR28 for their ability to confer resistance or sensitivity to p-F-Phe (see section (b), above). The results (not shown) demonstrated that pheS genes encoding the Gly295 mutant form (single or double exchanges) allowed (reduced) growth of transformed RR28 and growth was wild-type-like with the truncated M5 variant. This reflected the fact that those a subunits were inefficient or inactive in RR28, as already observed in strain KA2 (Figs 3, 4 and 5). In contrast, all mutants previously shown to possess a KAS-complementing pheS gene prevented growth of RR23 It was concluded, therefore, that they behave like the wild-type with respect to sensitivity to p-F-Phe by accepting the analogue as substrate in viva. These results were confirmed in vitro by the finding that the ratio of p-F-[‘4C]Phe/[‘4C]Phe incorporation into tRNA by the mutants corresponded to that for wild-type PheRS (data not shown). To further assess the substrate spectrum of the various mutants, a selection of phenylalanine analogues and other substrates was analysed in vitro for competition with [ 14C]Phe incorporation catalysed by the mutant PheRSs. The set of tested amino acids (either analysed singly or in pool groups) was identical to that listed below in Table 4. The competition tests yielded relative remaining [ 14C]Phe incorporation in the presence of a 50-fold excess of the L-enantiomer(s) of the potentially competing substance. From the results it emerged that the mutant a subunits did not reveal a significant shift in amino acid substrate specificity (data not shown). (h) Mutating

the alanine at position 294 affects Phe binding

Since an alanine-to-serine exchange at position 294 of a PheRS a subunit mutant was shown to be responsible for exclusion of the Phe analogue p-F-Phe from the enzymatic reaction, we rationalized that steric interactions might exist between residue 294 and the para-position of the aromatic ring of the substrate phenylalanine. To test this assumption directly Ala294 was replaced by smaller or larger amino acids. Figure 7(a) shows that residues with volumes larger than serine (Cys, Thr, Val) exhibited reduced


(a)

111

Synthetase SpecQicity

In who activity: complementation of KA2 at 40°C PBLS Gly Cys Val Phe Tyr

Postitiin295

Ml

NW Growth at 40°C

0

-

++

++

++

++

Trp stop WI ++

0

richmedium;

(b)

Initial velocities of aminoacylation at different temperatures

Aminoacylation activity (nmol Phe / min mg) 6 5 0

4

26°C 28°C. p.i

3 H 40°C 2

ffl 45°C

1 0

pBLS Gly* Cys Val* Phe Tyr Trp stop

0

(wt)

(C)

Approximate kinetic parameters at 28°C KM for Phe (PM)

Apparent Lt (l/min) la0 T

T8

Gly

Cys Val Phe Tyr Trp (W

Figure 5. Phenotypic effects of amino acid exchanges at position 295 of the PheRS CLsubunit. For description of the Figure see legend to Fig. 4. An asterisk next to the replaced amino acid (in (b)) indicates that the initial velocity of aminoacylation at 40°C and 45°C could not be determined accurately due to thermal denaturation during the assay. In these cases. the remaining aminoacylation velocity after 2 min was scored. KA2 complementation activities. The colonies containing the mutated gene for the Va1294 enzyme were tiny at 40°C and apparently dead because, when restreaked, they were unable to grow even at 30°C. In vitro assays confirmed the drop in PheRS

activity for the mutants Cys294, Thr294 and Va1294, which was most pronounced at high temperature (Fig. 7(b)). In addition, these three mutants showed a sharp decline in aminoacylation with time in vitro, but only at high temperatures

112

P. Ka.st and H. Hennecke

(a)

In who activity: complementation of KA2 at 40°C

Posilion2@3/zs6

pBlS Gly/Gly VaVGly GlyNal I 0J.M

GrcMhat40°C:

0

_

_

++

VaINal

++

Pha T#kp (wl) ++

++

richmedium; amgicillin

(b)

Initial vebcities of aminoacylation at different temperatures

Aminoacylation activity (nmol Phe / min mg)

6T 5 0 26°C 26%

p.i.

n 40% N 45°C

0 pBLS GlyKily Vaffily GlyNd’ VaINal*

(C)

phe Ml

TWW’

Approximate kinetic parameters at 28 “C

Apparent bt (l/min)

4 for Phe (PM)

160

12

140

10

120 100

6

H&al

6 q &l

80

60

4

40 2

20 0

0

GlylGly ValMy Figure 6. Phenotypic subunit. For description

GlyNal VaINal

Pha iti

Trplrrp

effects of simultaneous amino acid exchanges both at position of the Figure see legends to Figs 4 and 5.

(data not shown). This behaviour, already described for certain position 295 mutants (see section (f), above), points to reduced protein stability. In contrast, a decrease of aminoacylation with time was not observed with mutants Gly294 and Ser294 (data not shown). As shown in Figure 7(b), both mutants showed rather high activities at 28 “C (75 y.

293 and 295 of the PheRS u

of wild-type) and the values at high temperatures (40°C and 45°C) were only moderately reduced. Significant differences appeared when the kinetic parameters of the position 294 variants were determined (Fig. 7(c)). The binding affinity for phenylalanine was lowered in all residue 294 mutants, as indicated by increases in the apparent KM values


(a)

In wivo activity: complementation ~BLS Gly

Postitiin294

(V-4 Growth at 40%:

0

++

113


of KA2 at 40°C

Ala 0

Ser

Cys

Thr

Val

++

++

+

+

+

richmedium;

w

Initial velocities of aminoacylation temperatures

at different

Aminoacylation activity (nmol Phe / min mg) 6

cl 28°C 28% p.i.

n WC lsi 45°C

0

PBLS, Gly

(C)

Approximate

Ala (wtl

Ser

Cys* Thr’ Val*

kinetic parameters at 28°C 4 for Phe (@vt) 30 T

Apparent kcat (l/min) 250

T

Gly

Ala WI

Ser

Cys

Thr

Val

Figure 7. Phenotypic effects of amino acid exchanges at position 294 of the PheRS u subunit. The amino acid variants are arranged according to increasing volume (Chothia, 1984). For further description of the Figure see legends to Figs 4 and 5.

relative to the wild-type following defined as 190%). group of Ala294 resulted apparent reaction rate k,,,

Ala294 value (in the Removing the methyl in a reduction of the to 85O/o, whereas the

to 1640/;, (mutant apparent KM was increased Gly294). An increase in size of the side-chain of Ala294 to Ser also caused an increase in KM, even by a factor of 4. Although the k,,, of mutant Ser294


114 RatiopF-[14C]Phe I [W]Phaincwpmtim(+) and pF-Pheccmptedremaining [14C]Phe inmrporatii (A)

was

increased

to

142%,

the

catalytic

efficiency

(k,,JK,) was reduced to 34% of the wild-type level. For mutants Cys294, Thr294 and Va1294, catalytic efficiencies were lowered to 12 %, 18% and 16%, respectively, mainly due to small apparent k,,, values (Fig. 7(c)).

I\ I \ I \

(i) Position 294 is a PheRS specijicity determinant

w

70

80

80

loo

110

120

130

MO

The differential effects of position 294 mutants on phenylalanine binding confirmed the idea that this residue may be in contact with the amino acid substrate. To obtain more details about the interactions with the substrate, effects of other substrates and substrate analogues on activity of residue 294 mutants were tested in vitro and in vivo.

150

Aminoacidv&mmatpc#an 294[inAs]

Figure 8. Relative p-F-Phe/Phe specificities of position 294 PheRS mutants. The ratios of initial velocities in independent aminoacylation assays with p-F-[ 14C]Phe or [14C]Phe as substrates are displayed together with the remaining [14C]Phe incorporation in the presence of excess competing, unlabelled p-F-Phe (no competitor added equals 1). The ~says were carried out at 28°C. The values are plotted against the volume of the corresponding position 294 amino acid (Chothia, 1984). The wild-type enzyme (Ala294) is underlined.

(i) p-F-Phe incorporation and competition Initial velocities (at 28°C) of incorporation of [ ‘*C]Phe and p-F-[14C]Phe into tRNA were compared for the different position 294 mutants. The ratio of p-F-[14C]Phe/[‘4C]Phe incorporation (Fig. 8) revealed the following characteristics: mutant Gly294 (ratio p-F-Phe/Phe = 090) incorporated p-F-Phe essentially as well as the cognate substrate Phe, in contrast to the wild-type (Ala294; ratio = Q.53). As expected (section (b), above) the

Table 4 EJect of substrates and substrate analogues on position 294 mutant PheRS activities Wildtype

A. In viwo growth tests: mutant plasmids in strain RR28 (p-F-Phe resistant)t Minimal medium, Ala294 Thr294 37 “C Gly294 Ser294 Cys294 - p-F-Phe + p-F-Phe

++

++ 0

B. In vitro competition of Phe incorporation Ala294 Gly294 Substance

Ph4 TYr aa-pool 111 aa-pool 211 aa-pool 311 aa-pool 411 p-F-Phe m-F-Phe o-F-Phe 3-F-Tyr j-thien-2-ylalanine aa-pool 511 aa-pool 61)

6 93 94 101 97 100 26 53 71 105 57 97 85

vector

Position 294 mutant a subunits

7 53 89 91 88 94 8 34 63 89 69 20 41

++ +

++ +

++ f

Va1294 ++ +

by substrate analogues (in %)$ Thr294 Cys294 Ser294

Va1294

10 92 106 109 104 107 59 65 85 100 62 101 84

11 91 95 101 96 94 27 49 65 101 53 108 80

5 51 89 100 98 96 so 45 73 88 38 96 82

8 89 98 98 94 101 46 59 77 102 70 106 86

pBLS ++ +

tconditions for the growth tests were as described in Materials and Methods. The vector alone (pBLS) served as negative control. Growth of single colonies is specified from good growth to no growth at all, in the order: + + > + > + > - >O. f The competition assay was carried out as described in Materials and Methods with an estimated error of 10 to 15%. QIsotope dilution effect (positive control) by addition of a 50-fold excess of unlabelled Phe. I( The different pool groups contained the following components: aa-pool 1 (~-Asp, L-His, ~-Pro); aapool 2 (L-Arg, L-Asn, L-Glu, L-Gin, L-Lys); aa-pool 3 (L-Ile, L-Leu, L-Met, L-Trp, L-Val); aa-pool 4 (Gly, tAla, L-Cys, L-&x, L-Thr); aa-pool 5 @-Cl-Phe; p-Br-Phe, m-Cl-Phe); aa-pool 6 (2,3-di-fluoro-Phe; 2,4di-fluoro-Phe, 2,5-di-fluoro-Phe, 2,6-di-fluoro-Phe, 3,4-di-fluoro-Phe, 3.5di-fluoro-Phe). Each substance was present at a concentration of 1 rnM (of the L-enantiomer) in the competition tests.

Altered

Phenylalanyl-tRNA

mutant Ser294 did not incorporate p-F-Phe at a significant level (ratio I 604). Mutants Cys294 and, to a lesser extent, Thr294 showed intermediate ratios (634 and 0.40, respectively) that ranged between the wild-type (Ala294) and the p-F-Pheexcluding variant (Ser294); the differences to the wild-type, however, were at the limit of significance. With a ratio of 953, the Va1294 mutant exhibited again wild-type specificity (Fig. 8). This pattern of relative p-F-Phe/Phe specificity is exactly reflected by competition experiments in which [14C]Phe incorporation was competed by a 50-fold excess of p-F-Phe (Fig. 8, Table 4B). The largest competition appeared in the Gly294 mutant, which was in the range of the isotope dilution effect, i.e. the value obtained by adding the same concentration of unlabelled Phe to the assay (Table 4). In the Ser294 mutant, p-F-Phe did not significantly compete with Phe. Also for the other position 294 variants, the values of remaining Phe incorporation correlated well with the corresponding ratios of p-F-Phe/Phe aminoacylation (Fig. 8). These results were further confirmed by in vivo RR28 experiments (Table 4A). E. coli strain (p-F-Phe-resistant) showed some marginal growth on p-F-Phe-containing plates when a wild-type pheS gene (encoding the Ala294 variant) was present on a plasmid. Tn contrast, the presence of a Gly294 mutant a subunit prevented any trace of growth. This p-F-Phe “supersensitive” phenotype is explained by an enhanced detrimental p-F-Phe incorporation as compared to wild-type PheRS, which parallelled the in vitro results (see above). The intermediate growth of cells carrying genes encoding the Cys294 and Thr294 mutant forms could be rationalized by the partial exclusion of p-F-Phe from the enzymatic reaction (Fig. 8; Table 4B). An alternative explanation is that their PheRSs have essentially wild-type specificity but at a reduced activity level. This seems to be the case for the Va1294 enzyme (Table 4A and B). (ii) In vitro effects of other substrates and substrate analogues

Further in vitro competition tests were carried out with substances listed in Table 4B. To limit the number of assays, we pooled compounds believed to not compete with Phe in the wild-type PheRS reaction. When a pool exhibited competition with Phe in the test, with a particular mutant, individual members of this pool were then tested separately. Among the 19 non-cognate proteinogenic amino acids, only Tyr showed a slight tendency to compete with Phe. The wild-type PheRS exhibited about 90 to 95:/, remaining Phe incorporation (Table 4B), which is consistent with a previous report (Gabius et al., 1983a), in which activation and aminoacylation of Tyr by E. coli PheRS was quantified. Whereas the Cys294, Thr294 and Va1294 variants showed wild-type specificity with respect to tyrosine, this substance interfered substantially with aminoacylation by the Gly294 and the Ser294 mutants. The result with Tyr is parallelled by the slight

Synthetase

SpeciJicity

competition effect of 3-F-Tyr, which was observed exclusively with these two mutants (Table 4B). Other substrate analogues (besides the aforementioned p-F-Phe) that are known to exert their toxic action in vivo by being incorporated into cellular protein instead of Phe (Richmond, 1962; Shive & Skinner, 1963; Hortin & Boime, 1983) are m-F-Phe, o-F-Phe and j?-thien-2-ylalanine. Although t’he values for competition with [14C]Phe obtained for these analogues corresponded in general to the values expected from previous experiments with the wild-type enzyme (Hennecke & Bock, 1975; Gabius et al., 1983a), some variations between wild-type and mutant PheRSs were apparent (Table 4B). For instance, /I-thien-2-ylalanine (with its smaller ring than Phe) caused a significantly larger competition with the PheRS variant Ser294 than with the wildtype (Table 4B). By considering the marked differences in p-F-Phe competition between wild-type and Ser294 enzymes (Fig. 8; Table 4), we wished to explore the limits of substrate discrimination in both variants. Therefore, we analysed remaining r,-[‘4C]Phe incorporation in the presence of varying concentrations of either unlabelled L-Phe or unlabelled p-deutero-nPhe, which is only marginally smaller than Phe (a C-2H bond may be shorter by up to 0907 A (1 A = 61 nm) than a C-‘H bond; Bartell et al., 1961; Mitchell & Phillips, 1975). From the titration curves in these competition assays with the wild-type and the Ser294 enzymes (not shown), no differences were detectable between the two substrates within the accuracy of the experiment of (+5%). The most striking differential competition effect was found with pool groups 5 and 6, which significantly reduced [14C]Phe incorporation by the Gly294 enzyme (Table 4B). In contrast, for the wild-type and the other mutant PheRS variants, pool group 5 (p-Cl-Phe, m-Cl-Phe, p-Br-Phe) did not compete, and the large pool group 6 (containing ditluoro substituted phenylalanines) competed only slightly with the cognate substrate. Subsequently, each member of these pool groups was tested separately for its ability to compete with Phe. The results are summarized in Table 5. In contrast to the wild-type enzyme, which was not markedly inhibited by the substrate analogues, the addition of p-Cl-Phe and even p-Br-Phe caused a clear reduction of Phe incorporation catalysed by the Gly294 PheRS by more than 70 y0 and 5096, respectively Table 5). Difluoro-derivatives, having one substitution at the para-position, also showed increased competition with the cognate substrate in the Gly294 variant. The relaxed specificity of the Gly294 mutant appeared to be directed mainly to the para-position of the amino acid substrate; this was indicated, for instance, by the missing effect of m-Cl-Phe (Table 5). (iii) In vivo phenotypes on different

of PheRS mutants: growth Phe analogue-containing plates

It became of interest to test whether the ability of certain substrate analogues to compete with Phe is

116


Table 5 Competition eJfects by individual members of amino acid pool groups 5 and 6 on PheRS mutant Gly294 and the wild-type

with genes for Gly294 and Ser294 mutant a subunits (Table 6).

4. Discussion o/0 Remaining Phe incorporation in PheRS variant Competing substrate analogue None Phe para-chloro-Phet para-bromo-Phet meta-chloro-Phet 2,3-di-fluoro-Phe$ 2,4-di-fluoro-Phe$ 2,5-di-fluoro-PheJ 2,6-di-fluoro-Phe$ 3,4-di-fluoro-Phej 3,5-di-fluoro-PheS

Ala294 (wild-type)

Gly294

100 3 105 104 102 99 97 98 102 94 99

100 7 28 46 99 92 80 98 103 56 93

The competition tests were Materials and Methods. Each independent assays incubated for in the range of 5 %. t Member of pool group 5 (see $ Member of pool group 6 (see

(a) Prom a PheRS mutation to the Phe binding site?

carried out as described in number is the average of 4 5 min at 28°C. Deviations were Table 4). Table 4).

of significance in vivo. Therefore, the growth of E. coli strains carrying plasmids with wild-type and mutant pheS genes was analysed on different analogue-containing agar plates. The results in the p-F-Phe-resistant strain RR28 are shown in Table 6. An analogous growth pattern was also observed using the thermosensitive PheRS strain KA2 (data not shown). For the para-halogenated Phe derivatives, the results in vivo (Table 6) matched exactly those from competition experiments in vitro (Tables 4B and 5). The strain carrying wild-type pheS was sensitive only to p-F-Phe. If the Gly294 mutant gene was present, the strain showed extreme sensitivity not only to p-F-Phe but also to the p-Cl-Phe and p-Br-Phe analogues (Table 6). The addition of tyrosine to agar plates showed no growth inhibition, neither in cells carrying wild-type pheS, nor in those

5294, the PheRS a subunit mutation responsible for p-F-Phe resistance, was thought to point directly to amino acids in contact with the substrate Phe. This assumption was validated on a functional basis by site-directed mutagenesis experiments which brought additional evidence in favour of residue 294 as being involved in phenylalanine binding (see below). As shown in Figure 2, mutation S294 mapped within a PheRS-specific conserved region and, furthermore, within motif 3, one of the three conserved sequence motifs that define the class II aminoacyl-tRNA synthetases (Eriani et al., 1999a). We thus propose that motif 3 might constitute a component of the amino acid substrate binding domain in PheRS, and more generally, in aminoacyl-tRNA synthetases of class II. This proposal is supported by the crystal structure of SerRS (Cusack et al., 1990), the only class II tRNA synthetase structure published up to now; although the absence of substrates in the crystal did not permit a definitive identification of the binding sites, a large cavity within the globular domain of the enzyme was assumed to be the active site. The position in SerRS corresponding to the motif 3 residue 294 of the PheRS a subunit lies in a p sheet that builds the bottom of the putative active site. Of course, to extrapolate the assignment of structural elements from SerRS to specific positions in PheRS is only valid if the secondary and tertiary structure is as conserved among class II tRNA synthetases as it was found among class I members (Burbaum et al., 1999). The completion of the crystal structure of class II AspRS from yeast (complexed with tRNA) should make this clearer (Ruff et al., 1988; Cusack et al., 1990). At any rate, one should be aware that the situation with PheRS may be more complicated because the additional j? subunit (which shows no homology to other synthetases) seems to be involved in tRNA binding (Khodyreva et al., 1985:

Table 6 In vivo phenotypes of PheRS mutants grown in the presence of Phe analogues Amino acid at position 294 of a subunit, encoded by: Strain/plasmid RR28 RR28/pBLS RR28/pKSBl-WI RR28/pKSBl-M4G RR28/pKSBl-M4S

Chromosome

Plasmid

Ser Ser Ser Ser Ser

.~ Alaf G’Y Ser

Growth on supplemented minimal medium?, ampicillin, IPTG at 37°C with addition of (955 mM end concentration):

0 ++ ++ ++ ++

L-p-F-Phe

L-p-Cl-Phe

L-p-Br-Phe

L-Tyr

0 +

0 ++ ++ 0 ++

0 ++ ++ 0 ++

0 ++ ++ ++ ++

0 +

t Medium and conditions for the growth tests were described in Materials and Methods. Growth of single colonies is characterized good (+ +), normal (+), marginal (-) growth to no growth at all (0). $ Plasmid with wild-type pkS gene.

from

Altered Phenylalanyl-tRNA Hountondji et al., 1987; Sanni et al., 1991) and might contribute to the active site (see below; Baltzinger et al., 1979; Khodyreva et al., 1985). The functional importance of the conserved motif 3 was demonstrated by mutations in yeast and E. coli AspRSs (Prevost et al., 1989; Eriani et al., 1990b), in E. coli AsnRS (Anselme & Hgrtlein, 1991), in yeast mitochondrial LysRS (Gatti & Tzagoloff, 1991) and in yeast PheRS (Sanni et al., 1990), which drastically impaired enzyme activity. The results of this work further emphasized the importance of motif 3 in E. coli PheRS with the Phe295-to-Gly295 exchange, which led to a dramatic loss of activity in vitro and in vivo. From an increased &, value for ATP in AsnRS mutated in motif 3, Anselme & Hirtlein (1991) suggested that motif 3 might participate in ATP binding in class II tRNA synthetases. This would not exclude a simultaneous involvement of this sequence in amino acid substrate binding, since formation requires both aminoacyladenylate substrates to come close together, and their binding sites may therefore be immediately adjacent, as exemplified, for instance, for TyrRS (Brick et al., 1989). A participation of motif 3 in aminoacyladenylate synthesis was recently proposed on the basis of sequence similarities between AspRS and Asn synthetase (Gatti & Tzagoloff, 1991). Three-dimensional structural information is now needed to elucidate the exact role of motif 3 in PheRS. The generation of crystals of Thermus thermophilus PheRS (Chernaya et al., 1987; Ankilova et al., 1988) suitable for X-ray structural analysis was a promising step towards this goal, and, therefore, we are currently engaged in determining the nucleotide the sequence of T. thrrmophilus pheST genes. (b) A method for assaying mutated genes for e.rsential proteins at normal cellular levels The activitv of t)he mutant PheRS c1subunits was measured in k140 extracts of a host strain with a thermosensitive PheRS that carried the mutated pheS genes on a multicopy expression vector. The high gene dosage of plasmidial pheS, used to outcompete potential effects coming from the chromosomal copy, was shown not to influence the PheRS activity level. This was due to the normal expression of the chromosomally encoded b subunits (as shown by Western blots), which are needed to form functional tetrameric enzymes (Hennecke & B&k, 1975). Analogous results that showed expression of the pheST operon to be independent of the pheS gene dosage have been reported earlier (Springer et al.. 1983, 1985). The low amount of CI subunits seen in Western blots after overexpression of plasmid-encoded pheS genes might be explained by degradation of overproduced pheS gene products, once they are not complexed with p subunits. Free c( subunits might be recognized as abnormal cellular components and could induct, drhgradative processes (Goff &

Synthetase Speci$city

117

Goldberg, 1985). A low amount of PheRS M:subunit,, when expressed without #?,was already observed in earlier reports (Plumbridge et al., 1980; Plumbridge & Springer, 1982). With the exception of the inactive, truncated mutant form M5, which was rapidly degraded, all other mutants were able to compete with the chromosomally encoded a subunit in strain RR28 to engage with the p subunit in the formation of a holoenzyme. There was no correlation between aminoacylation activity and content of overproduced PheRS a subunit in S140 extracts. This indicates that the absence of excess free u subunits does not interfere with the determination of mutant PheRS activities, as long as the stoichiometric relation of a to /? subunits is not below one. An examination of background activities revealed that the host-encoded a2fi2 tetramers had no activity in vitro at 28 “C, the permissive temperature for growth, as already observed previously for the parent strain NP37 (Eidlic & Neidhardt, 1965; Neidhardt, 1966; B&k, 1968). The presence of a of hybrid tetramers considerable percentage (containing 1 chromosome and 1 plasmid-encoded c( subunit) could be ruled out from (1) thermoinactivation tests and (2) from the results of p-F-Phe incorporation and competition studies (e.g. with the p-F-PheR Ser294 a subunit: Hennecke, 1976). Although the influence of hybrid enzymes in cell extracts with comparably high aminoacylation activities may be neglected, a contribution to the low values of some thermosensitive mutants (e.g. those containing Gly295) cannot be excluded. Thus. the activit.ies obtained in those cases may br overestimates. The method described here for assaying in vitro mutagenized essential genes at the normal cellular level represents an easy alternative to the chromosomal integration of these mutant genes. The same strategy should be applicable to many multisubunit. enzymes (essential or non-essential ones) whose activity is strictly dependent on the presence of all subunits. A requirement for the system to work is that the overexpression of the mutated gene used t’o out-compete the resident gene product does not affect the synthesis of t.he other subunit,(s) encoded by the chromosome.

(c) The role of the aromatic residues at positiow and 295 in the a subunit

293

In this work, the two phenylalanine residues flanking position 294 of the PheRS c1subunit were replaced by other amino acids. From in vivo and in vitro results it became clear that aromatic interactions between the substrate phenylalanine and those residues are not essential for a.ctivity. Determination of apparent KM values and competition studies with substrate analogues suggested that the side-chains of the two residues 293 and 295 are not at all involved in Phe binding. A drastic thermosensitive behaviour was apparent with several PheRS mutants in ,vitro, indi-

118


eating a general destabilization of the protein structure (Griitter et al., 1979; Matthews et al., 1987; Menendez-Arias & Argos, 1989; Toth $ Schimmel, 1990). At elevated temperatures, the contribution of residue 295 to the stabilization of the protein structure was clearly more important than that of position 293. Residue 295 seems to maintain a stabilized structure via hydrophobic interactions: the hydrophobic Trp could fully replace Phe295, whereas more polar and/or smaller residues produced only unfavourable interactions. This agrees very well with the good conservation of this position in different organisms (Fig. 2), the only deviation from Phe being Trp in yeast cytoplasmic PheRS. A structural role for a conserved but non-essential aromatic residue in maintaining the conformation of epidermal growth factor was recently proposed by Engler et al. (1991). The less conserved position 293 in PheRS seems to tolerate polar residues (Tyr, Cys). Interestingly, replacing both Phe residues by Trp strongly reduced PheRS temperature stability, even though each substitution by itself had little detrimental effect. This may point to an interaction between the two positions 293 and 295. In the double mutant, the two bulky Trp residues might hinder each other in providing good stabilization to the enzyme and might even distort the conformation of the substrate binding pocket as reflected by the slightly increased KM value for Phe. The reversibly thermosensitive PheRS mutants constructed in this work may possibly be affected in the dissociation parameters of the subunits, as discussed for other thermosensitive mutant proteins (Jasin et al., 1985; Collier & Johnson, 1990), including the PheRS’” from strain NP37 (Bock, 1968; Comer & Bock, 1976). Residues 293 and 295 might thus be located close to one of the many subunit contact sites (see Fig. 1; Bock, 1968; Dessen et al., 1990) where they could interact with another subunit. A location of the presumed Phe binding site, residue 294, near a subunit interface would be in agreement with biochemical affinity labelling data. Using a Phe affinity label, Khodyreva et al. (1985) could locate the Phe binding site on the a subunit. However, when reactive ATP and aminoacyladenylate analogues were used, both a and b subunits were labelled. It was proposed that the corresponding binding sites (and therefore the catalytic site) of E. coli PheRS were close to or at the a/? subunit interface (Khodyreva et al., 1985; Hountondji et al., 1987). The same was postulated for yeast and turkey liver PheRS (Baltzinger et al., 1979; Renaud et al., -1982; Fasiolo et al., 1989; Gabius et al., 1983b) and, based on an analysis of a and /I subunit mutants, also for E. coli GlyRS (Toth & Schimmel, 1990). GlyRS, the other tRNA synthetase having an alp2 subunit structure, is immunologically related to E. coli PheRS (Nagel et al., 1988). Interestingly, sequence homologies between both enzymes are restricted to the presence of motif 3 in the small subunit and, hence, to the region analysed in this work.

(d) Changing the substrate spec@city by mutating Ala294 Mutagenizing position 294 produced the most interesting effects. From KM determinations and competition with substrate analogues it seemed obvious that the binding of the amino acid substrate was affected. Although variations in apparent KM values for the cognate substrate were only within one order of magnitude, they are comparable to the factor of 2 obtained after exchanging one of the specificity-determining residues in TyrRS (Tyr34 to Phe34; Fersht et al., 1985). With PheRS, wild-type-like activity was only obtained when the small amino acids Gly, Ala and Ser were present at position 294. However, a drop in activity was observed in vivo and in vitro, when the amino acid volume at position 294 was larger than Ser (Cys294, Thr294 and Va1294). It is possible that a critical size increase above Ser sterically alters the topology of the binding site and leads to a (reversible) destabilization of the protein structure, as indicated by the strongly thermosensitive behaviour of these mutants. In this context it should be recalled that a small amino acid is conserved at this position in different PheRSs (Fig. 2). Three position 294 mutations will be discussed in more detail in the following. (i) Ser at position 294: a model for the mechanism of p-F-Phe resistance The fact that in p-F-PheR strains the slightly bigger substrate analogue p-F-Phe is excluded from the enzymatic reaction due to an alanine-to-serine exchange led to further considerations concerning the mechanisms involved in specific substrate recognition. In Figure 9(a), we present a simple model that explains the exclusion of the substrate analogue mainly for steric reasons. The prinicpal assumption is that position 294 of the PheRS a subunit makes direct contact with the para-position of the aromatic ring of Phe. In wild-type PheRS, the Ala294 would thus allow tight fitting of the analogue (and the natural substrate) into the binding pocket (Fig. 9(a), centre). By contrast, Ser294 with its hydroxyl group would prevent a complete entering of p-F-Phe into the binding site due to steric hindrance, whereas Phe still fits. The OH-group might even establish a F-H hydrogen bond, thereby further displacing the analogue from the catalytically productive binding position (Fig. 9(a), right side). The question of whether the difference in van der Waals’ radii (Bondi, 1964) between H (1.20 A) and F (l-47 A) and the potential of the electronegative F to form hydrogen bonds are sufficient to explain the remains unanswered. Our observed phenotype, model does not account for the differences in electron distribution in the aromatic ring systems between p-F-Phe and Phe, although this could also be a cause for differentiation and discrimination. Previous binding studies with substrate analogues, however, indicated that steric effects are clearly


GIY*‘~

Ala2s4

G~Y*‘~ (WlM W)

Figure 9. Hypothetical model for interactions between amino acid substrates and position 294 residues in PheRS CIsubunit variants. (a) The substrate analogue p-F-Phe is arcommodated best in the binding cavity of the Gly294 variant, whereas Ser294 does not allow complete entering of p-F-Phe into the binding pocket. (b) The parahydroxyl group of the non-cognate amino acid Tyr prevents binding to the hydrophobic pocket of the wildtype PheRS. However, interactions of Tyr with the Gly294 and Ser294 variants may be possible due to an

cavity

and the potential

119

TyrRS (Fersht et al., 1985; Brick $ Blow, 1987; Freist & Sternbach, 1988). In the case of PheRS, it could be argued that for steric reasons an entering of Tyr into the binding pocket of the Ser294 variant is incomplete (Fig. 9(b)), thus leading to non-productive binding. This would explain the normal growth of cells producing Ser294 mutant PheRS on agar plates containing Tyr (which caused a cellular Tyr concentration at least 3 to 5 times that of Phe; Brown, 1970). Alternatively, the proofreading system of E. coli PheRS (Gabius et al., 1983aJ may have been able to cope with enhanced Tyr misact’ivation by the Ser294 mutant, and could therefore account for the absence of significant misacylation of tRNAPhe with Tyr, too.

(Wild type)

enlarged binding


to form

H-bonds. respectively.

dominant in determining amino acid specificity in the PheRS hydrophobic pocket (Santi 6 Danenberg, 1971). The KM increase for Phe in the Ser294 mutant, as compared to the wild-type, is also consistent with the model: the Ser residue might sterically interfere with optimal binding of phenylalanine. This is in agreement with the interpretation of results from subtilisin variant.s with Ala or Ser substitutions in t’he substrate specificity pocket (Estell et al., 1986). However, the binding affinity of PheRS for the slightly smaller p-deutero-Phe was, compared to Phe, not improved in the Ser294 mutant. This showed up the lower limit of the enzyme’s capability to significantly discriminate between steritally comparable substrates. In contrast to p-F-Phe, tyrosine significantly competed with the incorporation of the cognate substrate Phe by the Ser294 enzyme. A possible explanation is that Tyr forms H-bonds with the hydroxyl group of Ser294, thereby stabilizing the site binding of Tyr in the Phe recognition (Fig. 9(b)). H-bonding between enzyme residues and the para-hydroxyl group of Tyr is known to play an essential role in specific binding and recognition of the cognate substrate (and selection against Phe) in

(ii) Cys at position 294 The mutant Cys294 showed a tendency for exclusion of p-F-Phe. This phenotype corroborates that of the structurally similar Ser294 derivative and can be explained by a mechanism analogous to that proposed above. The introduced Cys residue could become useful for active site modifications with reactive substrate analogues (Khodyreva et al., 1985; Hountondji et al., 1987), especially with respect to proving any direct contacts between the para-position of the substrate’s aromatic ring and residue 294. Furthermore, once suitable crystals of E. coli PheRS are obtained, heavy metal atom derivatives at the Cys294 residue could greatly aid in the structural determination by X-ray diffraction analysis (Rould et aZ., 1989). (iii) Gly at position 294: a PheRS with relaxed substrate speci$city In line with the model proposed for the Ser294 mutant, one would predict the formation of an enlarged cavity for accommodating the puruposition of the aromatic ring of the amino acid substrate when Ala294 is replaced by a, Gly residue. As illustrated in Figure 9(b), a tyrosine substrate will not enter the Phe binding pocket in wild-type PheRS due to the lack of space for accommodation of the (possibly hydrated) p-hydroxyl group. A very similar situation of steric repulsion of Tyr was described by Estell et al. (1986). In contrast, Tyr is accommodated in the large cavity at the bottom of the presumptive binding pocket of the Gly294 variant (Fig. 9(b)), as experimentally confirmed by the enhanced competition of Phe aminoacylation by Tyr. That cells producing the Gly294 mutant exhibited no impaired growth on Tyr-containing plates could be explained by reasons already given for the Ser294 variant (see above). Incorporation and competition studies with parahalogenated analogues provided further evidence for an increased cavity for the para-position of the amino acid substrate in the Gly294 enzyme (see the model in Fig. 9(a)). Under our test conditions, the mutant enzyme, as opposed to the wild-type, had lost the ability to distinguish p-F-Phe from the natural substrate. Interestingly, the yeast cytoplasmic PheRS, which happens to have a Gly

120


residue at the corresponding sequence position (Fig. 2), also discriminates more poorly against p-F-Phe than the wild-type E. coli PheRS: the ratio (kcaJK&,c to (kcaJKM)p-F-Phcis 45 times higher for the E. COG enzyme (Gabius et al., 1983a). Furthermore, the spectrum of substrates for the Gly294 variant was extended to Phe analogues with para-substituents larger than fluorine (van der Waals’ radius: I*47 A; Bondi, 1964). Unlike the wild-type, mutant Gly294 allowed considerable competition of p-Cl-Phe and p-Br-Phe (van der Waals’ radii: Cl, 1.77 A; Br, 1.92 A) with Phe. In addition, those substances, like the para-fluorinated Phe, completely prevented growth of strains carrying the gene for mutant Gly294. It was clear that the para-halogenated Phe analogues had to exert their toxicity in vivo by interacting with the Gly294 mutant PheRS, an interaction already shown to occur in vitro (Table 5). For reasons of analogy to p-F-Phe and parallelism to other systems (e.g. incorporation of furanomycin by IleRS; Kohno et al., 1990), it seems highly probable that the growth inhibition by p-Cl-Phe and p-Br-Phe was caused by a detrimental incorporation of those analogues into cellular protein. However, protein incorporation studies in vivo (Fangman & Neidhardt, 1964; Koide et al., 1988) or in vitro (Kohno et al., 1990) will be necessary to support this contention (Hortin & Boime, 1983). Furthermore, radiolabelled analogues (as in the case of p-F-Phe) and purified Gly294 PheRS will be useful tools in future work to definitively prove the incorporation of those analogues into tRNAphe (Igloi et al., 1979). A change in substrate specificity created by protein engineering and/or chemical modification was achieved for the proteases subtilisin (Estell et al., 1986), trypsin (Craik et al., 1985) and carboxypeptidase Y (Beth t Breddam, 1988). Similar to the position 294 PheRS mutants, it emerged from these studies that substrate specificity was largely determined by steric complementarity. Even in the synthetases aminoacyl-tRNA “superspecific” (Bosshard, 1976), amino acid specificity could be altered by exchanging binding pocket residues. The substitution of a residue that determines specificity for Tyr through hydrogen bonding to the substrate’s para-hydroxyl group (Brick & Blow, 1987) lowered the discrimination of Phe relative to Tyr in the activation reaction by one order of magnitude (Fersht et al., 1985). Yet the biological effect may be small because the relative specificity for activation of the cognate substrate compared to Phe was still higher by a factor of lo4 in the mutant enzyme. In addition, Tyr specificity might be further enhanced in the transfer reaction to tRNATy’ by proofreading mechanisms (Freist & Sternbach, 1988; Freist, 1989). The substrate specificity mutant Gly294 (and also Ser294), however, showed a very distinct phenotype with some non-proteinogenic substrate analogues in the in vivo growth tests. Apparently, the proofreading system of PheRS (Gabius et al., 1983a), which may have been optimized solely to discriminate against non-cognate substrates that

occur naturally in the cell (Wilson & Hatfield, 1984), did not prevent incorporation of xenobiotic substances. The enlarged binding cavity in the Gly294 enzyme that accounts for the accommodation of large paru-substituents in the Phe analogues opens up a new perspective for the in vivo synthesis of polypeptides containing non-proteinogenic amino acids. The Gly294 mutant PheRS may be used, for instance, to incorporate a variety of Phe analogues into proteins of pharmaceutical interest such as polypeptide growth factors (James & Bradshaw, 1984) or endocrine peptide hormones (Douglass et al., 1984; Lynch & Snyder, 1986), which may result in artificial polypeptides with novel pharmacologically interesting properties. We are currently exploring ways to establish an in vivo system for the incorporation of amino acid analogues into target proteins with the help of our PheRS mutants. Different approaches for incorporating nonproteinogenic amino acids into protein were tested out previously (Morris & Schlesinger, 1972; Sykes et al., 1974; Lu et al., 1976; Pines et al., 1981; Koide et al., 1988; Kohno et al., 1990). However, these authors worked with wild-type enzymes that use only a limited spectrum of amino acid analogues as substrates (Hortin & Boime, 1983). Our examples with the PheRS mutants Ser294 and Gly294 demonstrate that aminoacyl-tRNA synthetases can be specifically “tailored” by mutation to use substrates of interest. We are most grateful to Claudia Wehrli for her expert technical assistance throughout the whole work. We thank Barbara Keller for cloning the pheS gene from S. typhimurium. We are greatly indebted to S. Cantoreggi and D. Seebach for the chemical synthesis of several Phe analogues used in this work and for the suggestion to test p-deutero-Phe. We also thank P. Babczinski for sending us other Phe derivatives, H. Sternbach for providing purified PheRS, T. Schmidheini for fast protein liquid oligonucleotides, purification of chromatography A. Birkmann for a Plkc lysate, A. Bock for p-F-[14C]Phe and K. Girgenrath and L. Diirselen for scintillation counting. This work was supported by a grant from the Federal Institute of Technology Zurich.

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The substrate binding site specificity of a modified tryptophan-synthetase beta2 subunit from Escherichia coli.

A single amino acid substitution affects the substrate specificity of the seryl-tRNA synthetase homologue.

Synchronization of cell division in Escherichia coli by amino acid starvation: strain specificity.

Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity.

Escherichia coli thymidylate synthase: amino acid substitutions by suppression of amber nonsense mutations.

Derepressed levels of glutamate synthase and glutamine synthetase in Escherichia coli mutants altered in glutamyl-transfer ribonucleic acid synthetase.

Examination of polypeptide substrate specificity for Escherichia coli ClpB.

Protease II from Escherichia coli. Substrate specificity and kinetic properties.

Escherichia coli DNA polymerases II and III: substrate specificity.

Control of acetohydroxy acid synthetase in Escherichia coli 9723.

Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Escherichia coli by pseudomonic acid.

Escherichia coli mutants with altered ribosomal ribonucleic acid metabolism.

The amino acid sequence of beta-galactosidase of Escherichia coli.

Control of Escherichia coli lysyl-tRNA synthetase expression by anaerobiosis.

Regulation of branched-chain amino acid transport in Escherichia coli.

The glutamyl-tRNA synthetase of Escherichia coli: substrate-induced protection against its thermal inactivation.

Crystals of glutamine synthetase from Escherichia coli.

EntG activity of Escherichia coli enterobactin synthetase.

2'-Deoxy-2'-fluorouridine-5'-phosphate: an alternative substrate for thymidylate synthetase from Escherichia coli K12.

Amino-terminal sequences of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium.

Structural and mutational analysis of amino acid residues involved in ATP specificity of Escherichia coli acetate kinase.

Enhanced biosynthesis of 3,4-dihydroxybutyric acid by engineered Escherichia coli in a dual-substrate system.

Cloning and characterization of the Escherichia coli Heptosyltransferase III: Exploring substrate specificity in lipopolysaccharide core biosynthesis.

Allosteric Control of Substrate Specificity of the Escherichia coli ADP-Glucose Pyrophosphorylase.