Appl Microbiol Biotechnol(1990) 33:553-559

Applied AFtcrobiology Biotechnology © Springer-Verlag 1990

Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila Ignacio Prieto ~, Julio Martin 2, Roberto Arche 2, Piedad Fern~ndez 1, Augustin P6rez-Aranda ~, and Jose Luis Barbero I 1 Departamentode Investigaci6n,Antibi6ticos Farina, S. A., Antonio L6pez 109, 28026 Madrid, Spain 2 Departamento de Bioquimicay Biologia Molecular I, Universidad Complutense de Madrid, Facultad de Ciencias Quimicas, 28040 Madrid, Spain Received 17 November 1989/Accepted 6 April 1990

Summary. Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl4-aminobenzoic acid and the catalytic constant kca t for phenylacetyl-4-aminobenzoic acid. Likewise, the phenylmethylsulphonyl-fluoride sensitivity was significantly decreased. It is concluded that the 168 residue is involved in binding by interaction with the acid moiety of the substrate. A putative penicillin-binding domain was located in penicillin acylase by sequence homology with other penicillin-recognizing enzymes. Lys374 and His481, the conserved amino acid residues that are essential for catalysis in these enzymes, can be changed in penicillin acylase with no changes to the kcat and phenylmethylsulphonyl fluoride reactivity, but change the Kin. The likelihood of the existence of this proposed penicillin binding site is discussed. The reported results might be used to alter the substrate specificity of penicillin acylase in order to hydrolyse substrates of industrial significance other than penicillins.

Introduction The systematic alteration of the sequence of a protein by site-directed mutagenesis permits study of the involvement of particular amino acids in the structure and/or activity of an enzyme. This technique has been used to introduce specific base changes in the penicillin G acylase gene of Kluyvera citrophila ATCC 21285. This enzyme (EC 3.5.1.11, PA) is industrially used to hydrolyse penicillin G (Pen G) to phenylacetic acid and 6-aminopenicillanic acid (6-APA), the major intermediate in the production of semi-synthetic penicillins (Mahajan 1984). PA has two different subunits which

Offprint requests to: I. Prieto

are obtained from a single precursor protein and both subunits are required for activity (Daumy et al. 1985). The mature protein has great conformational stability, which accords with the high beta content of the structure as determined by circular dichroism studies (Mfirquez et al. 1988). Although the substrate specificity (Margolin et al. 1980) and the kinetic mechanism (Konecny et al. 1983) of PA has been extensively investigated, the molecular basis of substrate binding and catalysis, as well as the tridimentional structure of the protein remain to be determined. Nevertheless, it has been established that the catalytic mechanism progresses through an acyl-enzyme intermediate (Konecny et al. 1983). Using site-directed reagents the existence of an essential serine residue which could be directly involved in the binding of the acyl moiety of the substrate (Siewinski et al. 1984) has also been demonstrated. In previous work, we have cloned, sequenced and overexpressed in Escherichia coli the PA gene from K. citrophila under its own promoter control (Garcia and Buesa 1986; Barbero et al. 1986) allowing manipulation of the gene and producing mutant proteins. The sequence of K. citrophila PA precursor is 87% homologous with the sequence of the E. coli precursor (Schureacher et al. 1986). Since the tridimentional structure of the protein is not known, the criteria to design mutant proteins must be based on other structural or functional evidence. Williams and Zuzel (1985) showed that mutations in Met168 of PA from E. coli produced changes in substrate specificity. Moreover, Oliver et al. (1985) have proposed an amino acid homology between E. coli penicillin binding proteins (PBPs) and a region around Met168 in the small subunit of K. citrophila PA. Assuming functional homology for this residue in E. coli and K. citrophila enzymes, we have mutated Met168 in PA from the latter. Also, up to seven regions of sequence homology among several active-site-serine penicillinrecognizing enzymes have been identified (Joris et al. 1988). Based on these homologies, we have identified a putative penicillin binding site in the beta subunit of

554 P A f r o m K. citrophila. M u t a t i o n s o f this site s h o u l d p e r m i t i d e n t i f i c a t i o n o f t h e e s s e n t i a l s e r i n e a n d to o b s e r v e t h e effect o f a l t e r a t i o n s o n t h e s u b s t r a t e specificity.

M a t e r i a l s and methods

Bacterial strains, phages and plasmids. The bacterial strains used in this work were E. eoli ATCC 11105. E. coli JMI03 A(lac pro), thi, strA, supE, endA, sbcB, hsdR- F'tra D36, proAB, lacI q, ZAM15) (Messing 1983) and E, eoli HB101 F', hsdS20 (ry, my), recA13, ara-14, proA2, lacY1, galK2, rpsL20 (Smr), xyl-5, mtl-1, supE44, ~,- (Boyer and Roulland-Dussoix 1969). Bacteriophage M13mp10 was obtained from Amersham Searle (Buckinghamshire, UK). The plasmid pYKD59 used in this study was obtained by a Pvu-I-SalI deletion of the pYKH5 plasmid (Gareia and Buesa 1986) and it contains a Cm r and the PA gene of K. eitrophila under the control of its own promoter. D NA manipulations, oligonueleotide-directed mutagenesis and synthesis of the organic compounds. Restriction enzymes were obtained from Amersham Searle. Both T4 DNA polymerase and T4 DNA ligase were from Bio-Rad Laboratories (Richmond, Calif, USA). Transformation of competent cells was performed according to Hanahan (1983). Plasmid DNA was prepared according to the sodium dodecyl sulphate (SDS)-NaOH lysis procedure of Maniatis et al. (1982). Oligonucleotides 30-33 bases long were synthesized using phosphoramidite chemistry (Beaucage and Caruthers 1981) on an Applied Biosystems (Foster City, Calif., USA) DNA synthesizer, model 380A. The oligonucleotides were purified by gel filtration and preparative polyacrylamide gel electrophoresis (PAGE). The construction of the PA mutants was done using the protocols of the Muta-Gene in vitro mutagenesis kit of Bio-Rad Laboratories (Bio-Rad 1987), Phenylacetyl-4-aminobenzoic acid (PAPABA) was prepared as described by Szewczuk et al. (1980). Pen G and 6-APA were supplied by Antibirticos (Leon, Spain) Penicillin V (Pen V), fluorescamine and p-dimethylaminobenzaldehyde were purchased from Sigma (St. Louis, Mo, USA) Phenylmethylsulphonyl fluoride (PMSF) and other analytical grade reagents were from Merck (Darmstadt, FRG). Protein purification. The PA from E. coil and mutants of K. eitrophila enzyme were purified as previously described for the wildtype enzyme of Kluyvera (Barbero et al. 1986). The activity of the enzymes was determined by colorimetric methods using 6-APA (Balasingham et al. 1972) and 4-aminobenzoic derivatives (Szewczuk et al. 1980) as substrates. Purity was checked by SDA-PAGE following the method of Laemmli (1970) using Coomassie Blue as a stain.

Protein determination. The content of protein in the samples was assayed by the method of Bardford (1976) during the purification procedure and by amino acid analysis and spectroscopic methods when pure. Amino acid analysis was performed by standard protein hydrolysis and quantitation of amino acids in a Durrum D-500 analyser (Dionex, Sunnyvale, Calif, USA). The molar concentration of enzyme was calculated taking into account the amino acid composition from the nucleotide sequence (Barbero et al. 1986). Spectroscopic determination of the concentration of protein was performed using an E°'81o/"=2.24 calculated by the method of Wetlaufer (1962). The concentrations calculated by both methods agreed. Kinetic measures and active site titration. The reaction was measured by titrating with fluorescamine the 6-APA released by PA, following the procedure of Baker (1983). Fluorescence was measured at 475 nm after 390 nm excitation in a Perkin-Elmer (Nor-

walk, Conn, USA) MPF-44E fluroescence spectrophotometer thermostatted at 25 ° C. Initial rates were calculated by least-squares regression of the time progress curves and the K,, and the catalytic constant (kcat), were calculated by fitting the data to the Woolf equation. At least eigth different substrate concentrations were assayed in duplicate. The molar concentration of the active sites of PA was determined by reaction with PMSF (Siewinski et al. 1984). After preincubation with inhibitor, the PA activity of each sample was measured by quantitation of the 6-APA released with p-dimethylaminobenzaldehyde (PDAB; Balasingham et al. 1972).

Results

Olioonucleotide-directed site-specific mutagenesis W e h a v e c a r r i e d o u t m u t a g e n e s i s in t w o r e g i o n s o f t h e P A gene, p r e s e n t in t h e p Y K D 5 9 p l a s m i d , t r y i n g to change the amino acid residues with a small distortion in t h e s e c o n d a r y s t r u c t u r e o f t h e p r o t e i n .

Alpha subunit. By c o m p a r i s o n o f t h e a m i n o a c i d seq u e n c e s o f P A f r o m E. coli w i t h t h o s e o f o t h e r PBPs, O l i v e r et al. (1985) s u g g e s t e d t h a t t h e r e g i o n b e t w e e n M e t 168 a n d Lys 191 c o u l d b e i n v o l v e d in t h e b i n d i n g o f t h e P e n G m o l e c u l e . T h e a m i n o a c i d s e q u e n c e o f this r e g i o n is also h i g h l y c o n s e r v e d in t h e P A f r o m K. citrophila ( B a r b e r o et al. 1986). W i l l i a m s a n d Z u z e l (1985) h a v e also d e s c r i b e d m u t a n t s in M e t 168 o f P A f r o m E. coli A T C C 9637 w h i c h a r e m o d i f i e d in s u b s t r a t e specificity. W e h a v e c h a n g e d the A T G c o d o n o f t h e e q u i v a l ent a m i n o a c i d in t h e P A o f K. citrophila g e n e to c o d o n s G C G ( A l a 168) o r G T G (Val 168), l o s i n g a n NcoI res t r i c t i o n site p l a c e d in p o s i t i o n 500 o f t h e c o d i f y i n g s t r a n d o f t h e P A g e n e ( B a r b e r o et al. 1986). U s i n g t h e m e t h o d o f G a r n i e r et al. (1978) f o r t h e prediction of protein secondary structure we have obs e r v e d n o c h a n g e in t h e A l a 168 m u t a n t a n d a c h a n g e f r o m a l p h a h e l i x to b e t a s h e e t in t h e Val 168 m u t a n t (Fig. 1). W e h a v e o b t a i n e d a n o t h e r m u t a n t in this a r e a b y c h a n g i n g t h e t r i p l e t G G C o f G l y 166 to G A C ( A s p 166), w h i c h g e n e r a t e s n e w AccI, H i n d l I a n d S a l I res t r i c t i o n t a r g e t s a n d has t h e effect o f e x t e n d i n g t h e carb o x y - e n d a l p h a h e l i x in w h i c h M e t 168 is i n c l u d e d . MUTANT WT

SEQUENCE

STRUCTURE

GCGATGATTTTTGTCGGCACCATGGCGAACCGGTTTT

~uuu_ua_uuc~tccccc~ ~(~c~tccccc~

ALA

168

....... TTTTTGTCGGCACCGCGGCGAACCGGTTTT

VAL

168

....... TTTTTGTCGGCACCGTGGCGAACCGGTTTT

~ f l ~ C c c c c ~

ASP

166

GCGATGATTTTTGTCGACACCATGGCGAACCGG

UUUUUUUuuutCCCCC~

....

Fig. 1. Oligonucleotides used and secondary structure predictions for the alpha subunit mutants of penicillin acylase (PA). The wildtype sequence of nucleotides 481 to 517 of the codifying strand of the PA gene is compared with sequences of the oligonucleotides used for obtaining the mutants. The wild-type Gly I66 and Met 168 codons and the destroyed and created restriction sites are underlined. The secondary structure prediction of the 162 to 179 amino acid zone of the PA wild-type protein is compared with the equivalent positions in the mutant molecules. Gly 166 and Met 168 positions in the wild-type are underlined: a, alpha helix; fl, beta sheet; t, beta turn; c, random coil

555 This mutation has been carried out because the mutants described by Williams and Zuzel (1985) that disturb this helix produce less active proteins.

have replaced Ser 372 for Thr 372, but were not able to obtain any detectable mutant protein.

Kinetic equations Beta subunit. The group of penicillin-recognizing proteins may be members of a single superfamily of activesite-serine enzymes that have several conserved boxes which consist o f strictly identical or homologous amino acids (Joris et al. 1988). Daumy et al. (1985) showed that the essential active serine residue is located in the beta subunit o f PA from Proteus rettgeri. Because of the similar subunit composition of PA from P. rettgeri and K. citrophila, we have only sought the sequence Ser-XX-Lys proposed by Joris et al. (1988) for box II in the beta-subunit (Fig. 2A). There are five possible box II sites containing the catalytic serine, but only one is in agreement with the location of the other conserved boxes. We have focused our attention in the II and VII boxes, the most conserved among the group of penicillin-recognizing proteins. In box II we have changed the AAG Lys 375 codon to AAC Asn 375 codon, introducing new XrnaI and Sinai restriction sites. This change produces disruption of the carboxy-end of the box-IIcontaining alpha helix and increases the probability of the beta turn placed behind this helix (Fig. 2B). The beta turn probability calculated by the method of Chou and Fasman (1979) rises from the 3.93 value of the wild-type to 11.51 for this mutant. We have also changed the His 481 CAT codon in box VII to Tyr 481 TAT codon, producing a new AccI site and increasing, in the place where this box is located, the probability of a beta sheet structure from the 0.910 estimated value for wild-type to the 1.060 value for mutant. Finally, we

A. BoxI boxII TISWGSTAGFGDDVDIFAEKLSAEKPGYYQHNGEWVKMLSRKETIAVKDG cct~cccttcca~uauuuuuuu~utt@@~ccc~aa~@~tcc

iii

boxIII boxIV QPETFTVWRTLDGNVIKTDTRTQTAYAKARAWAG_KEVASLLAWTHQMKAK c c ~ t c t t c ~ = ~ ~ e t c c

161

boxV boxVI boxVII NWPEWTQQAAKQALTINWYYADVNGNIGYV~TGAYPDRQP ccc~u~u~uu~ttt~@~@~t~ctt~@t

From the following acyl-enzyme based mechanism '

where E, S, E', P~ and P,2 are enzyme, substrate, acylenzyme, and products respectively and K~ are velocity constants. The steady-state kinetic parameters for PA activity are k2k3 . k3 (k_ ~+ k2). kcat kik2 kcat = k2+k3 ' g m = k~(k2+k3) ' Km - k_~+k~ [1]. The temperature dependence of Km and k~at for penicillin G hydrolysis by the wild-type PA (Martin et al. 1990) indicates that k_~ is greater than k2 and no rate-limiting step exists in the mechanism. Assuming these results, Eq. 1 can be simplified to

k~k3 ; Km kcat -- k2 -1- k3

Ksk3 . kcat k2. being Ks = k_~ -- ke + k~' K m = ~ ' k~

the dissociation constant of the E : S complex. The k~at values provides information about the enzyme acylation and deacylation processes. Expressed in terms of transition state theory, the equilibrium constant between E + S and the transition state ES* is proportional to the activation free energy (AG*) of k c a t / g m , so that kca t

Km-

kT [-AG* I h e x p \ R T ]"

where K is the Boltzmann constant, R is the gas constant and T is the absolute temperature.

201

B.

MUTANT

SEQUENCE WT AACTTTCCGCCGAGAA_~GCCGGGCTATTACC

STRUCTURE ~ a t t ~ c c c ~ a ~

ASN 375 MUT A A C T T T C C G C C G A G A A C C C G G G C T A T T A C C WT A T A T C G G C T A T G T G C A T A C C G G C G C C T A T C

~a~aaaaccttt~ccca~uu ~ttt~St~ctt~ttc

T Y R 481 MUT A T A T C G G C T A T G T G T A T A C C G G C G C C T A T C

~ t t ~ 8 ~ t t ~ t t c

Fig. 2. A Location of the I-Vn conserved amino acid boxes proposed by Joris et al. (1988) in the primary sequence of PA protein from Kluyvera citrophila and its secondary structure prediction. The numbers indicate the amino acid position in the mature beta subunit. The amino acid composition of each box is underlined. B Oligonucleotides used and secondary structure predictions for the beta subunit PA mutants, Nucleotides 1109 to 1138 (Asn 375 mutant) and 1427 to 1456 (Tyr 481 mutant) of the wild-type are compared with the sequences of the oligonucleotides used for obtaining the mutants. The underlined sequences and the abbreviations are as in Fig. 1

Fig. 3. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis of purified PA proteins. In each well --~20 ~g of purified PA protein was subjected to electrophoresis: 1, Sigma mol. wt. SDS-70L markers with their mol. wt. in daltons × 10-3; 2, PA from K. citrophila; 3, PA from E. coli; 4, Ala 168 PA mutant; 5, Val 168 PA mutant; 6, Asp 166 PA mutant; 7, Tyr 481 PA mutant; 8, Asn 375 PA mutant

556

In this sense, from analysis of the variation o f AG~ produced by either site-directed mutations in the enzyme or structural changes in the substrate, the effect of these events on the stabilization o f ES* can be deduced (Wilkinson et al. 1983).

Effect in the k~,,, of the acid and amino components of the substrate We have purified the five mutant proteins, the wildtype enzyme and the E. coli ATCC 11105 PA (Fig. 3) and we have used purified fractions to obtain the kinetic constants for Pen G, PAPABA and Pen V. In all the assayed proteins, the lowest kc~, was for Pen V and the kca t for Pen G was higher than for PAPABA (Fig. 4). Nevertheless, the structural differences between Pen G and Pen V are smaller than between Pen G and PAPABA. This suggests that in the acylation and deacylation steps of the enzyme the structure o f the acid moiety o f the substrate is more determinant than the amine moiety. Since k3 is the same for Pen G and PAPABA, the interaction between the amine moiety and the enzyme should directly affect the ke value.

Kinetic parameters of mutants in the alpha subunit of the PA protein The comparison of kinetic parameters for mutant PAs with respect to the wild-type is shown in Fig. 4. The percentages in relation to the wild-type o f the k~at values for Ala 168 and Val 168 mutants are not the same for Pen G and PAPABA. Since the acyl-enzyme intermediate from both substrates is identical, the k3 should be the same. Therefore, the 168 residue could be involved in the acylation kinetic step defined by k2. On the other hand, significant differences in kca t between both mutants only exist for PAPABA. Because the amine moiety of the substrate is 6-APA in Pen G and Pen V but 4-aminobenzoic acid (PABA) in PAPABA, it is suggested that the 168 residue could be located in a reALA

VAL

ASP

ASN

PEN G

TYR

ALA

VAL

ASP

ASN

"rYR

gion that interacts with the substrate depending on whether the amine component is 6-APA or PABA. From the kcat values of the wild-type enzyme, it is derived that smaller free energy o f activation for the acyl-enzyme formation is required for Pen G than for PAPABA. The Ala 168 and Val 168 mutants produce an increase in the difference between both activation energies. Since the changes in kcat/grn for Pen G and PAPABA are similar for the two mutants, it indicates that a selective stabilization of the E:S complex is occurring with PAPABA as substrate. From these results it might be concluded that the Met 168 residue is located in a domain o f direct interaction with the amine moiety of the substrate. Nevertheless, we can only affirm that this region interacts with the substrate in a different way depending on the structure of the amine moiety. Then, an inadequate binding o f the substrate in the active site could unstabilise another part of the molecule that interacts directly with the 168 residue. In fact, the different behaviour o f these mutants in their reaction with P M S F (see below) and the increase in the k3 of the Ala 168 mutant with Pen G (Martin et al. 1990) indicate that the effect o f the mutation is on the acylation step. The Asp 166 mutant also shows a different kinetic behaviour with Pen G, Pen V or PAPABA. Pen G and PAPABA have similar k2 percentages with respect to the wild-type, but Pen V has a smaller k2 value when compared to the wild-type. Nevertheless, no difference between mutant and wild-type is observed. The kc,t value suggests that the mutation affects the acid part o f the substrate, whereas kcat/Km differences affect the amine moiety, indicating that the mutation should be located in a region of the protein in which the interaction with the substrate depends on both parts of it.

Kinetic parameters of mutants in the beta subunit of the PA protein The Asn 375 mutation does not alter the kat values (Fig. 4), but the k~,t/Km with Pen G and Pen V shows a desALA

VAL

ASP

ASN

TYR

PAPABA

Fig. 4. Comparison of kinetic parameters from wild-type and mutant penicillin acylases from K. citrophila. The corresponding enzymes and substrates are indicated respectively at the top and the bottom: ALA, Ala 168 mutant; VAL, Val 168 mutant; ASP, Asp 166 mutant; A SN, Asn 375 mutant; TYR, Tyr 481 mutant. Km(IxM)and I I I (kcat)(s-1) values of wild-type enzyme for each substrate were respectively: 18, 56.5 for penicillin G (PEN G); 137, 20.8 for phenylacetyl-4-aminobenzoic acid (PAPABA) and 47, 4.7 for penicillin V (PEN V). kcat mut Open bars, Ln (1/Km)mut , diagonal striped bars, Ln--;----~.; shaded bars, Ln (k~at/Km)mut, where (1/Km)wt (k~at/gm)wt /cc,twt mut = mutant and wt = wild-type

557 tabilization of the transition state with respect to the wild-type. However, the opposite behaviour has been found with PAPABA. The interaction between the 375 residue and the substrate is also modified in both the E : S complex and transition state. These results suggest that the Lys 375 residue, which has an important role in the penicillin-recognizing enzymes, has lost its essential catalytic function in PA but might still interact with the amine moiety of the substrate. The Tyr 481 mutation does not alter the kca t values but the stabilization of the transition state depends on this residue as can be inferred from the kcat/Km values obtained. Since the mutants were constructed on the basis of the sequence homology proposed by Joris et al. (1988), in which this residue is located in the beta-lactam binding domain, and due to the fact that kcat is not altered, the interaction may be with a common structural part of the amine moiety of the three substrates, i.e. the carboxylate group. The E. coli PA enzyme increased its efficiency for PAPABA. The sequences of E. coli and K. citrophila reA

veal that there are changes near the 372 serine residue in which some basic amino acids are substituted by acid residues. Nevertheless, the importance of these differences remain unascertained. Reaction with P M S F When the reaction of the wild-type and mutant enzymes with PMSF is compared, the mutants in the beta subunit behave in a similar way to the wild-type, i.e. the inhibition is linear and the intersection with the abscissa corresponds to the molar concentration of the active sites (Fig. 5A). However, the inhibition is not linear for alpha subunit mutants, indicating that either PMSF turnover is significant or the enzyme is able to react with more than one PMSF molecule. Therefore, the PMSF assay cannot be used as an active site titration method for this group of enzymes. This evidence sup: port the conclusion that the mutations in the alpha subunit have a large influence on the structural elements responsible for the binding by interaction with the acid moiety of the substrate. Mutations in the beta subunit should be located in the region of interaction with the amine moiety which is not present in the PMSF molecule.

1,S Discussion

,~~

0

,.n'E 0.5

J

~

B

100

PMSF (nM)

20O

3O0

4OO

.~0

Fig. 5 A, B. Active site titration of PA by reaction with phenylme-

thylsulphonyl fluoride. A Wild-type (t), Tyr 481 (@), and Ash 375 (A) enzymes from K. citrophila and wild-type enzyme from Escherichia coli (•). The active site concentration was calculated by extrapolation of the straight segment (broken line). B Val 168 (O), Asp 166 (@) and Ala 168 (A) PA mutants from K. citrophila. Broken lines represent the behaviour to be expected according to the active site concentration spectrophotometricallydetermined

The substitution of Met168 of PA from K. citrophila produced mutant enzymes with different catalytic properties than the wild-type enzyme. The results suggest that the 168 residue might be directly involved in the interaction with the acid moiety of the substrate. However, this interaction is also dependent on the amine moiety (6-APA or PABA) to be released. It should be pointed out that the conformational restrictions that are imposed by the rings of beta-lactam and PABA do not permit superposition of the two ends of Pen G and PAPABA molecules (phenyl and free carboxyl groups) in stereochemically identical positions. Therefore, the binding of the two moieties of the substrate are influencing each other, On the other hand, secondary structure prediction for the Val168 mutant (Fig. 1) shows a significant change from alpha helix to beta sheet. The effect on catalysis of this structural change should also be considered. It is worth nothing that the sequence comparison between PA from E. coli (Schumacher et al. 1986), cephalosporin acylase (Matsuda et al. 1987) and glutaryl-7-amino-cephalosporanic acid acylase from Pseudomonas (Matsuda et al. 1985) reveals a homology in a region preceding Met168, suggesting that this conserved domain might play the same role in recognizing a common substrate structure in all these proteins. Joris et al. (1988) reported that the more conserved sequence within PBPs and beta-lactamases is Ser-X-XLys, the serine residue being the essential active serine that is involved in the formation of the acyl-enzyme intermediate. In addition to this homology, other sequence boxes have been shown to be conserved in all

558 PA KC

SAEKPGYYQHNGEWVK

PBP IA

SNIKPFLYTAAMDKGL

PBP IB

SLAKPATYLTALSQPK

PBP 3

STVKPMVVMTALQRGV

PBP 5

SLTKMMTSYVIGQAMK

Fig. 6. Comparison of sequences of different penicillin binding proteins (PBP) from E. eoli close to the catalytic serine (underlined) and the corresponding sequence close to Ser 372 (underlined) of K. citrophila. The PBP sequences are taken from Keck et al. (1985). Bold letters represent the conserved amino acids among the different proteins compared according to Oliver et al. (1985) these proteins. A putative penicillin binding site has been f o u n d in the PA sequence in accordance with the conditions of Joris et al. (1988) and where the essential serine corresponds to Ser372 of the beta subunit in PA f r o m K. citrophila. It is necessary to point out that Oliver et al. (1985) suggested a sequence h o m o l o g y between PA and PBPs in a different region that included the 168 residue in the alpha subunit. However, the sequence Ser-X-X-Lys in PBPs was aligned with Ser-XX-Asp in PA and a deletion o f four residues between Met168 and Set177 was p r o p o s e d (Oliver et al. 1985). Furthermore, the Ser372 region we p r o p o s e shows homology with PBPs 1A, 1B, 3 and 5 f r o m E. coli that can be considered similar to that p r o p o s e d by Oliver et al. (1985) (Fig. 6). To evaluate the likelihood of our assumption, site-directed mutagenesis o f the most conserved residues was performed. The fact that Asn375 and Tyr481 mutations do not drastically decrease kcat points to a different location to the essential serine in PA than that expected from sequence comparison. Nevertheless, the possibility that Ser372 is the essential serine cannot be discarded. It has been previously p r o p o s e d that the lysine residue next to the active serine could participate in catalysis by the f o r m a t i o n o f a salt bridge with the carboxyl group of the substrate (Varetto et al. 1987) or b y assisting proton transfer f r o m the catalytic serine to the nitrogen a t o m o f the hydrolysable amide b o n d (Herzberg and Moult 1987). Since PA hydrolyses a different amide b o n d from the beta-lactamases, the role o f Lys375 in the catalytic steps should be less important, whereas the maintenance o f interactions m a y still have an important role in binding. In this sense, the Asn375 mutant should only cause a slight distortion of these interactions. On the other hand, the basic residue in the b o x VII o f Joris et al. (1988) seems to be involved in the stabilisation o f the negative charge of the carboxyl group of substrate (Herzberg and Moult 1987). The increase in kcat o f the Tyr481 mutant for all three substrates assayed might be in agreement with a decrease in the binding energy of the amine product when c o m p a r e d with the His481 wild-type enzyme. The existence of a c o m m o n penicillin binding domain in all the penicillin-recognizing enzymes, i.e. PBPs beta-lactamases and PAs, will not be unequivocally proved until the tridimensional structure of all these proteins is available. However, the results shown

in this work suggest that the residues within this domain that are essential in PBPs and lactamases do not play an important role in the chemical processes catalysed by PA, although their participation in substrate binding is maintained. Thus, evolution m a y have favoured the a p p e a r a n c e of other catalytic domains in PA that should be located in the proximity of the different amide bonds to be hydrolysed. It should be mentioned that the d o m a i n near the Met168 residue, which is conserved in different acylases and is involved in the binding of the acid moiety, m i g h t be of great importance within this context. The location o f the active serine residue in PA that directly participates in the formation o f the acyl-enzyme intermediate remain to be ascertained and is very valuable in order to prove the present conclusions. The results presented here contribute to a better understanding o f the molecular basis of the m e c h a n i s m of the PA reaction, with the final aim of producing engineered enzymes which could hydrolyse new beta-lactam substrates. Acknowledgements. We wish to thank Dr. E. Arribas of Antibi6ticos Farrna S. A. for synthesis of the oligonucleotides used in obtaining the PA protein mutants. We are also very grateful for the technical assistance of Dr. J. P6rez-Gil and J. M. Manchefio, from the Departamento de Bioquimica y Biologia Molecular I of the Universidad Complutense de Madrid.

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