Vol. 173, No. 6
JOURNAL OF BACTERIOLOGY, Mar. 1991, p. 1886-1893
0021-9193/91/061886-08$02.00/0 Copyright ) 1991, American Society for Microbiology
Relative Activities and Stabilities of Mutant Escherichia coli Tryptophan Synthase cx Subunits WOON KI LIM,t HAE JA SHIN,* DEBRA L. MILTON,§ AND JOHN K. HARDMAN*
Cellular and Developmental Molecular Biology Section, Department University of Alabama, Tuscaloosa, Alabama 35487
of Biology,
Received 10 October 1990/Accepted 2 January 1991
In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase a subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant a subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability- properties in crude extracts. With the exception of mutant a subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant a subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant a subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specffic mutagenesis of the Salmonella typhimurium tryptophan synthetase a subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant a subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the 02 subunit. Also among the mutant a subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the a polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth requirements of a trpA mutant host. Heat stability and potential folding-rate alterations are found in both enzymatically active and defective mutant a subunits. Tyr-4, Pro-28, Ser-33, Gly-44, Asp-46, Arg-89, Pro-96, and Cys-118 may be important for these properties, especially for folding. Two regions, one near Thr-24 and another near Met-101, have been also tentatively identified as important for increasing stability.
conformational changes between the subunits, whereby the rates of the a and 1 reactions are strongly enhanced by, respectively, the P2 and a subunits (i.e., by the a2132 complex). Similar subunit structures are found in most bacterial TSases. The a2P2 crystal structure has been recently solved for the Salmonella typhimurium enzyme (8). The sequences of the TSase polypeptides from E. coli and S. typhimurium are homologous (85% of the residue sites are identical; this is 93% if conservative differences are discounted), identity and the enzymes share virtually identical enzymatic properties. The three-dimensional structure on the S. typhimurium a2P2 complex is compatible with the enzymatic and structural solution studies of the E. coli enzyme. For example, there is an active site pit on the a subunit wherein indole 3-glycerol phosphate binds, and there is a clear channel between this site and the pyridoxal phosphate-containing site on the P2 subunit (3, 15, 26, 30). The availability of this structure has allowed more detailed interpretation of the roles of certain residues of the E. coli TSase identified as critical to activity and stability. Extensive early in vivo mutagenesis studies by Yanofsky and colleagues (2, 7, 20, 28, 29) of the E. coli trpA gene, which encodes the a subunit, utilized functional selective procedures and has yielded defective a subunits altered at only eight different residue sites in this 268-residue protein ly Leu-177, Gly(namely, residues Phe-22, Glu-49, Tyr-175, Lu-17, 211, Gly-213, Ser-234, and Gly-235). More recently (23), residue Asp-60 has been identified from this early mutant collection as critical.
Microbial tryptophan synthases (TSases) catalyze the terminal steps in tryptophan biosynthesis. Three reactions are catalyzed: indole-3-glycerol phosphate = indole + (a reaction) D-glyceraldehyde-3-phosphate (,B reaction) L-tryptophan indole-3-glycerol phosphate + L-serine -- D-glyceraldehyde(a13 reaction) 3-phosphate + L-tryptOphan The Escherichia coli enzyme has been the most extensively studied (for reviews, see references 16 and 27) and consists of an a subunit that can catalyze the a reaction and a pyridoxal phosphate-containing P2 subunit which can catalyze the P reaction. The physiological a2%32 complex alone catalyzes the ac reaction, which proceeds at two independent a,B sites via catalysis of the a reaction on the a subunit component, channeling the product (indole) to the pyridoxal phosphate site on the ,B component, where, in the presence of L-serine, it is converted to tryptophan. There is apparent subunit communication mediated by transduced indole + L-serine
->
* Corresponding Corresponding author. t Present address: Clinical Endocrinology Branch, National Institutes Health, stitutes Bethesdal MD 20892. of of Health, t Present address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294. § Present address: Cellular and Molecular Biology Unit, University of Umea, S-901 Umea, Sweden.
20892.9,Tyr175 Bethesda,reides 1886
VOL. 173, 1991
MUTANT E. COLI TRYPTOPHAN SYNTHASE ox SUBUNITS
In an effort to dissect more completely the roles of the residues in this protein in its functional, structural, and folding properties, researchers in this laboratory (18) have employed the random, saturation in vitro mutagenesis protocol described by Myers et al. (21), which includes a selection procedure based not on function but only on the fact that there was a DNA alteration on the trpA gene. In addition, we have constructed several overexpression vectors containing the trpA gene (wild type or mutant) to obtain the a subunits in sufficient yield for analysis. We report here an expansion of the initial studies to describe the in vivo behavior and general enzymatic and stability properties in crude extracts of 66 mutant a subunits singly altered at 49 different residue sites within the first 121 residues (ca. 45%) of the a polypeptide. (Some of this work was done in partial fulfillment of the Ph.D. requirements for W.K.L., Department of Biology, University of Alabama, Tuscaloosa.)
MATERIALS AND METHODS Bacterial strains, phage strains, and plasmids. E. coli RB797 (F' lacIq proL8larg Nalr Rif' recA sup lac pro XIII) served as the host for expression vectors for crude extract preparations of wild-type and most mutant a subunits. Strains MC1061 and LE392F' were used as described before (18) for the in vitro mutagenesis and sequencing procedures. trpA mutant strains A33 and A14-17 (both are recA and contain a missense and a double-nonsense mutation, respectively) served as host strains for certain plasmids that expressed low levels of soluble mutant a subunits and for determining the nutritional needs of a number of mutant a
subunits. Plasmids
pGC1, pGC2, pGCtrpAIF, pMHtrpAIR, pMH trpAIIF, pGCtrpAIIR, pGCtrpAIIIF, pGCtrpAIIIR, ptactrp AITc, and ptactrpAll, were utilized in the mutagenesis procedure and have been described before (18). Plasmids ptactrpA and pCATtrpA served as overexpression vectors for both wild-type and mutant at subunit production; each contains the trpA gene under control of the tac promoter and is inducible by lactose or isopropylthio-13-D-galactoside. Plasmid pCATtrpA contains the chloramphenicol acetyltransferase gene from pBR329, the replication origin from pBR322 and the tac promoter trpA gene from ptactrpA. Plasmid pCATtrpB, identical to pCATtrpA except that it contains the trpB gene, was employed for 12 subunit purification. Plasmid pCATtrpAIIIF(pBR) is a modified expression vector for trpA fragment III, employed for subcloning mutant trpA fragments III, and is identical to pCATtrpA except that a 1,840-bp sequence from pBR322 replaces trpA fragment III. Its use during the mutagenesis procedure is analogous to that for the modified expression vectors for trpA fragments I and II (18). Enzymes and chemicals. All reagents and enzymes used for the mutagenesis and enzyme analyses have been described before (18). The 12 subunit was purified as follows. A 1-liter batch of TYS medium (tryptone-yeast extract-NaCl; percentage ratio, 1:0.5:0.5) containing chloramphenicol (10O g/ ml) was inoculated with 20 ml of a similar overnight culture of RB797 containing pCATtrpB, grown to the early log phase (optical density at 550 nm, 0.4 to 0.5), induced with lactose (1%, final concentration), and then grown for an additional 4 to 5 h. Cells were harvested by centrifugation at 28,000 x g for 15 min, washed with 100 ml of 200 mM potassium phosphate buffer (pH 7.5) containing 5 mM EDTA-0.5 mM phenylmethylsufonyl fluoride-10 mM 1-mercaptoethanol-
1887
0.02 mM pyridoxal phosphate, suspended in 13 ml of this buffer, and sonicated. The suspension was centrifuged at 48,000 x g for 20 min. The supernatant solution was adjusted to 0.12 mM pyridoxal phosphate and heated at 63°C for 8 min, quickly cooled, centrifuged at 48,000 x g for 10 min and dialyzed for 16 h at 5°C against 110 volumes of the original buffer. The preparation was applied to a DEAE-Sephadex column (2.5 by 40 cm, equilibrated with the original buffer). The 12 subunit was eluted with a linear gradient (600 ml of original buffer and 600 ml of 0.5 M potassium phosphate buffer [pH 7.8] containing the above additions) at a flow rate of 16 ml/h. Fractions containing the electrophoretically homogeneous 2 subunit were combined and stored at -70°C. The specific activity of 12 subunit preparations was 2,600 U/mg. The at subunit was purified as described previously (18). Mutagenesis. The mutagenesis procedure described before (18) was used with the following modifications. After the single-stranded mutagenesis vector was subjected to chemical mutagenesis, it was annealed to a single-stranded backbone obtained from digests of the double-stranded homolog by restriction endonucleases B and C, and the mutagenized single-stranded trpA fragment sequences were copied with avian myeloblastosis virus reverse transcriptase. This fully double-stranded plasmid was used to transform strain MC1061. In addition, the preparative urea-formamide gradient gels employed for isolation of mutant trpA fragments II and III contained denaturant gradient concentrations of 65 to 90% and 52.5 to 77.5%, respectively. Preparation of crude extracts of a subunits. For the wildtype a subunit and the majority of the mutant a subunits, the encoding overexpression plasmids were contained in RB797. trpA 14-17 was employed as the host for expression vectors encoding mutant a subunits YC4, AV18, SL33, AV43, GS44, DG46, DN46, CR81, RC89, RL89, PL96, and CR118. For mutant at subunits with alterations at residue sites 1 through 66, the ptactrpA expression vector was employed; for the others, pCATtrpA was used. Overnight TYS (plus the appropriate antibiotic) cultures of RB797 containing the expression vectors were inoculated (0.6 to 30 ml of fresh TYS-antibiotic medium for RB797 or 4 to 200 ml of fresh TYS-antibiotic medium for trpA 14-17), grown for 1.5 to 2 h, induced with lactose (1%, final concentration), and grown for an additional 12 to 13 h for RB797 or 4 h for trpA 14-17. Cells were harvested by centrifugation at 28,000 x g for 10 min, suspended in 1.5 ml of 10 mM potassium phosphate buffer (pH 7.8) containing 5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol. The suspension was sonicated and centrifuged at 48,000 x g for 20 min. The supernatant solution (crude extract) was stored at -800C. Enzyme assays. The a subunit-specific reaction (a reaction) was assayed by a modification of the Smith and Yanofsky method (24). The reaction mixture (250 ,ul) included 0.18 M NaCl, 0.2 mM dithiothreitol, 0.05 mM pyridoxal phosphate, and 4 mM EDTA in addition to original constituents. When NH2OH was added, pyridoxal phosphate was omitted. After 20 min of incubation at 37°C, 50 ,ul of 1 M NaOH and 3 ml of water-saturated ethyl acetate were added, and the preparation was vigorously mixed. Aliquots (1.5 ml) of the cleared ethyl acetate layer were added to 2 ml of Ehrlich reagent, and the A540 was recorded. The 2subunit specific reaction (1 reaction) was assayed by the Kirschner et al. method (9); the overall a2132 subunit-specific reaction (ao3 reaction) was assayed by the Smith and Yanofsky method (24). Relative specific activities of wild-type and
1888
J. BACTERIOL.
LIM ET AL.
tt
tttt tt
t
tt
t
1.
ttt
1tt
7OKU14 LL2
LL
FIG.
t
Identity and distribution of amino acid substitutions in mutant
cx
subunits. The secondary structures for the S. typhimurium a
subunit for the mutagenized N-terminal, 121-residue region are shown along with turn (t) residues. Differences from the E. ccli enzyme are found at positions 6 (N-S), 12 (N-K), 13 (D-E), 15
(R->K),
42 (D-E), 52 (V-I), 68 (N-T), 90 (E-Q), 109 (N-K), 113 (A-*E), 117
(R-Q), and 120 (Q-K). Mutant designations are, for example, YC4, a Tyr-Cys change at position 4. Not listed above are the following mUltiPlY altered mUtant aL SUbUnitS: EGS/EG31, PS21/PL28, AV18/PL21, PT21/IV36, AT47/ATS9, GDS1/DN6O, PHS3/LQS8, GD75/AT86, TI77/YC102, AT74/FS114, GD75/DN112, TA77/HN92, QR8O/AV86, PS78IYC115, AT73/LP85, TA94/AV116, AV74/PS78, FS82/DN112, GD11O/MI1O1, LP85/AV103, FL72/GD98, LS11O/CR118, GS75/MI101, AT43/GSS1/ITS2, AV79/IV97/YC1O2, AT79/AT103/VA121, and
IV97/YC102/AV116.
mutant a subunits in crude extracts were measured in terms of pure 2 subunit with an excess of the a subunit. To insure that P2 (or a) subunit was added in excess, especially with crude extracts, a linear increase of activity with increased amounts of other subunit was monitored. Heat inactivation. The crude extracts (1 mg/ml) of at subunit in 0.1 M potassium phosphate (pH 7.8) containing 1 mM EDTA and 0.1 mM phenylmethylsufonyl fluoride were incubated at 52°C for 20 min. After treatment, they were cooled quickly on ice and assayed. The interval between heat treatment and enzymatic assay was kept constant for all proteins as a precaution to avoid the possibility that the activity of heat-treated a subunits may recover to some extent after heat treatment and during the assay. p-Reaction activity was utilized for several reasons. It is the most reliable among the three TSase reactions and circumvents major problems whereby some mutant a subunits exhibited varied levels of material in soluble form (12) and/or little or no detectable activities in the other TSase reactions. Calculation of relative amount of a subunit in crude extracts. The activity of each mutant a subunit was measured with various amounts of crude extracts in the ,B reaction in the presence of an excess of pure P2 subunit and calculated as units of au activity per milligram of pure 12 subunit. From the linear portion of this curve, the maximum activity can be determined, and comparison of these values with that of purified wild-type a subunit under similar conditions yields the relative maximum specific activity of the mutant a subunits. When the maximum specific activity of pure wildtype a subunit (units per milligram of a subunit) in the 1 reaction is known, the corresponding maximum specific activity of each mutant a subunit in this reaction can be estimated. Thus, the relative amounts of both wild-type and mutant a subunits (i.e., milligrams of a polypeptide per milligram of total soluble protein can be determined for any a subunit-containing crude extract. Solvent accessibility. The static solvent accessibility of residue in the X-ray crystal structure was calculated by the ACCESS program of Lee and Richards (10). Since only the structure of the a2P2 complex was known, the coordinates used for the a subunit were those remaining after the coordinates of 12 subunit, pyridoxal phosphate, and indole3-propanol phosphate were removed from the whole complex. The static accessibility of an amino-acid residue, X, in model tripeptides Ala-X-Ala as calculated in Lee and Richards (10) was used here as the reference accessibility of a fully extended polypeptide. When an amino acid had two
sets of data from different conformations, the average was adopted, and the solvent accessibility of Glu, Asp, and the a carbon of Gly was considered as those of Gln, Asn, and the side chain of Gly, respectively. Protein assays. The concentrations of purified a and 32 subunits were estimated from the extinction values (E278nm1% of 4.4 for the wild-type a subunit [1] and 6.5 for the 132 subunit [4]). The protein concentrations of crude extracts were measured by the microbiuret method (11). RESULTS AND DISCUSSION Altered residue sites in the mutant a subunits. The a subunit component of the a2132 TSase complex consists of alternating a helix and ,B-sheet structures in a a1-barrel motif (8). The trpA mutations identified in this study resulted in a subunits altered at residue sites within approximately the first half (residues 1 through 121) of the a polypeptide. Figure 1 shows the secondary structures within this region, the sites and identity of the single substitutions, and a list of multiple amino acid substitutions of mutant a subunits. A total of 64 residue sites have been altered in both ordered and random sequences. There are 49 different residue sites that have been singly altered with multiple amino acid substitutions at 16 of these sites. Thus, 66 mutant a subunits are now available containing single amino acid differences from the wild-type a subunit. As noted above, this region of the a polypeptide contains only two of the missense residue sites (Phe-22 and Glu-49) found in early random in vivo mutagenesis studies. All of the mutant trpA genes encoding these proteins are contained in overexpression vectors, which allow the expression of mutant a subunits'for study. As yet, none of mutant trpA genes encoding for the multiply altered a subunits has been subcloned into overexpression vectors. Instead we have focused our efforts here on describing initially the enzymatic and stability properties in crude extracts of the singly altered proteins. Enzymatic activities of mutant a subunits. The activity of each of the singly altered at subunits was assayed in crude extracts in the presence of a fixed amount of pure 132 subunit in the three TSase reactions (Table 1). Two broad classes of mutant a subunits can be seen; those (48 of 66) with normal activities and those (18 of 66) with altered activities in one or more of the reactions. Some general features of these results are worth considering at this point. First, as observed previously (18), about 25% of single-base-pair alterations lead to no amino acid
VOL. 173,
MUTANT E. COLI TRYPTOPHAN SYNTHASE at SUBUNITS
1991
1889
TABLE 1. In vitro and in vivo properties of mutant a subunitsa Mutants with defective activities
Relative sp act (%) in the following reaction:
Growth on minimal
EXPOSED 100
Defective in a and ot, reactions FS22
EG49 DN60 DG60 YC4 TK63 QR65 CR81 RL89
Defective in ,3 and ao reactions GD51 DG56
YC102 NS104 Defective in all TSase reactions PL28 PH53 PT53 AV67 CR118
REACTION
a
mediumb
44 0
JOURNAL OF BACTERIOLOGY, Mar. 1991, p. 1886-1893
0021-9193/91/061886-08$02.00/0 Copyright ) 1991, American Society for Microbiology
Relative Activities and Stabilities of Mutant Escherichia coli Tryptophan Synthase cx Subunits WOON KI LIM,t HAE JA SHIN,* DEBRA L. MILTON,§ AND JOHN K. HARDMAN*
Cellular and Developmental Molecular Biology Section, Department University of Alabama, Tuscaloosa, Alabama 35487
of Biology,
Received 10 October 1990/Accepted 2 January 1991
In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase a subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant a subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability- properties in crude extracts. With the exception of mutant a subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant a subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant a subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specffic mutagenesis of the Salmonella typhimurium tryptophan synthetase a subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant a subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the 02 subunit. Also among the mutant a subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the a polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth requirements of a trpA mutant host. Heat stability and potential folding-rate alterations are found in both enzymatically active and defective mutant a subunits. Tyr-4, Pro-28, Ser-33, Gly-44, Asp-46, Arg-89, Pro-96, and Cys-118 may be important for these properties, especially for folding. Two regions, one near Thr-24 and another near Met-101, have been also tentatively identified as important for increasing stability.
conformational changes between the subunits, whereby the rates of the a and 1 reactions are strongly enhanced by, respectively, the P2 and a subunits (i.e., by the a2132 complex). Similar subunit structures are found in most bacterial TSases. The a2P2 crystal structure has been recently solved for the Salmonella typhimurium enzyme (8). The sequences of the TSase polypeptides from E. coli and S. typhimurium are homologous (85% of the residue sites are identical; this is 93% if conservative differences are discounted), identity and the enzymes share virtually identical enzymatic properties. The three-dimensional structure on the S. typhimurium a2P2 complex is compatible with the enzymatic and structural solution studies of the E. coli enzyme. For example, there is an active site pit on the a subunit wherein indole 3-glycerol phosphate binds, and there is a clear channel between this site and the pyridoxal phosphate-containing site on the P2 subunit (3, 15, 26, 30). The availability of this structure has allowed more detailed interpretation of the roles of certain residues of the E. coli TSase identified as critical to activity and stability. Extensive early in vivo mutagenesis studies by Yanofsky and colleagues (2, 7, 20, 28, 29) of the E. coli trpA gene, which encodes the a subunit, utilized functional selective procedures and has yielded defective a subunits altered at only eight different residue sites in this 268-residue protein ly Leu-177, Gly(namely, residues Phe-22, Glu-49, Tyr-175, Lu-17, 211, Gly-213, Ser-234, and Gly-235). More recently (23), residue Asp-60 has been identified from this early mutant collection as critical.
Microbial tryptophan synthases (TSases) catalyze the terminal steps in tryptophan biosynthesis. Three reactions are catalyzed: indole-3-glycerol phosphate = indole + (a reaction) D-glyceraldehyde-3-phosphate (,B reaction) L-tryptophan indole-3-glycerol phosphate + L-serine -- D-glyceraldehyde(a13 reaction) 3-phosphate + L-tryptOphan The Escherichia coli enzyme has been the most extensively studied (for reviews, see references 16 and 27) and consists of an a subunit that can catalyze the a reaction and a pyridoxal phosphate-containing P2 subunit which can catalyze the P reaction. The physiological a2%32 complex alone catalyzes the ac reaction, which proceeds at two independent a,B sites via catalysis of the a reaction on the a subunit component, channeling the product (indole) to the pyridoxal phosphate site on the ,B component, where, in the presence of L-serine, it is converted to tryptophan. There is apparent subunit communication mediated by transduced indole + L-serine
->
* Corresponding Corresponding author. t Present address: Clinical Endocrinology Branch, National Institutes Health, stitutes Bethesdal MD 20892. of of Health, t Present address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294. § Present address: Cellular and Molecular Biology Unit, University of Umea, S-901 Umea, Sweden.
20892.9,Tyr175 Bethesda,reides 1886
VOL. 173, 1991
MUTANT E. COLI TRYPTOPHAN SYNTHASE ox SUBUNITS
In an effort to dissect more completely the roles of the residues in this protein in its functional, structural, and folding properties, researchers in this laboratory (18) have employed the random, saturation in vitro mutagenesis protocol described by Myers et al. (21), which includes a selection procedure based not on function but only on the fact that there was a DNA alteration on the trpA gene. In addition, we have constructed several overexpression vectors containing the trpA gene (wild type or mutant) to obtain the a subunits in sufficient yield for analysis. We report here an expansion of the initial studies to describe the in vivo behavior and general enzymatic and stability properties in crude extracts of 66 mutant a subunits singly altered at 49 different residue sites within the first 121 residues (ca. 45%) of the a polypeptide. (Some of this work was done in partial fulfillment of the Ph.D. requirements for W.K.L., Department of Biology, University of Alabama, Tuscaloosa.)
MATERIALS AND METHODS Bacterial strains, phage strains, and plasmids. E. coli RB797 (F' lacIq proL8larg Nalr Rif' recA sup lac pro XIII) served as the host for expression vectors for crude extract preparations of wild-type and most mutant a subunits. Strains MC1061 and LE392F' were used as described before (18) for the in vitro mutagenesis and sequencing procedures. trpA mutant strains A33 and A14-17 (both are recA and contain a missense and a double-nonsense mutation, respectively) served as host strains for certain plasmids that expressed low levels of soluble mutant a subunits and for determining the nutritional needs of a number of mutant a
subunits. Plasmids
pGC1, pGC2, pGCtrpAIF, pMHtrpAIR, pMH trpAIIF, pGCtrpAIIR, pGCtrpAIIIF, pGCtrpAIIIR, ptactrp AITc, and ptactrpAll, were utilized in the mutagenesis procedure and have been described before (18). Plasmids ptactrpA and pCATtrpA served as overexpression vectors for both wild-type and mutant at subunit production; each contains the trpA gene under control of the tac promoter and is inducible by lactose or isopropylthio-13-D-galactoside. Plasmid pCATtrpA contains the chloramphenicol acetyltransferase gene from pBR329, the replication origin from pBR322 and the tac promoter trpA gene from ptactrpA. Plasmid pCATtrpB, identical to pCATtrpA except that it contains the trpB gene, was employed for 12 subunit purification. Plasmid pCATtrpAIIIF(pBR) is a modified expression vector for trpA fragment III, employed for subcloning mutant trpA fragments III, and is identical to pCATtrpA except that a 1,840-bp sequence from pBR322 replaces trpA fragment III. Its use during the mutagenesis procedure is analogous to that for the modified expression vectors for trpA fragments I and II (18). Enzymes and chemicals. All reagents and enzymes used for the mutagenesis and enzyme analyses have been described before (18). The 12 subunit was purified as follows. A 1-liter batch of TYS medium (tryptone-yeast extract-NaCl; percentage ratio, 1:0.5:0.5) containing chloramphenicol (10O g/ ml) was inoculated with 20 ml of a similar overnight culture of RB797 containing pCATtrpB, grown to the early log phase (optical density at 550 nm, 0.4 to 0.5), induced with lactose (1%, final concentration), and then grown for an additional 4 to 5 h. Cells were harvested by centrifugation at 28,000 x g for 15 min, washed with 100 ml of 200 mM potassium phosphate buffer (pH 7.5) containing 5 mM EDTA-0.5 mM phenylmethylsufonyl fluoride-10 mM 1-mercaptoethanol-
1887
0.02 mM pyridoxal phosphate, suspended in 13 ml of this buffer, and sonicated. The suspension was centrifuged at 48,000 x g for 20 min. The supernatant solution was adjusted to 0.12 mM pyridoxal phosphate and heated at 63°C for 8 min, quickly cooled, centrifuged at 48,000 x g for 10 min and dialyzed for 16 h at 5°C against 110 volumes of the original buffer. The preparation was applied to a DEAE-Sephadex column (2.5 by 40 cm, equilibrated with the original buffer). The 12 subunit was eluted with a linear gradient (600 ml of original buffer and 600 ml of 0.5 M potassium phosphate buffer [pH 7.8] containing the above additions) at a flow rate of 16 ml/h. Fractions containing the electrophoretically homogeneous 2 subunit were combined and stored at -70°C. The specific activity of 12 subunit preparations was 2,600 U/mg. The at subunit was purified as described previously (18). Mutagenesis. The mutagenesis procedure described before (18) was used with the following modifications. After the single-stranded mutagenesis vector was subjected to chemical mutagenesis, it was annealed to a single-stranded backbone obtained from digests of the double-stranded homolog by restriction endonucleases B and C, and the mutagenized single-stranded trpA fragment sequences were copied with avian myeloblastosis virus reverse transcriptase. This fully double-stranded plasmid was used to transform strain MC1061. In addition, the preparative urea-formamide gradient gels employed for isolation of mutant trpA fragments II and III contained denaturant gradient concentrations of 65 to 90% and 52.5 to 77.5%, respectively. Preparation of crude extracts of a subunits. For the wildtype a subunit and the majority of the mutant a subunits, the encoding overexpression plasmids were contained in RB797. trpA 14-17 was employed as the host for expression vectors encoding mutant a subunits YC4, AV18, SL33, AV43, GS44, DG46, DN46, CR81, RC89, RL89, PL96, and CR118. For mutant at subunits with alterations at residue sites 1 through 66, the ptactrpA expression vector was employed; for the others, pCATtrpA was used. Overnight TYS (plus the appropriate antibiotic) cultures of RB797 containing the expression vectors were inoculated (0.6 to 30 ml of fresh TYS-antibiotic medium for RB797 or 4 to 200 ml of fresh TYS-antibiotic medium for trpA 14-17), grown for 1.5 to 2 h, induced with lactose (1%, final concentration), and grown for an additional 12 to 13 h for RB797 or 4 h for trpA 14-17. Cells were harvested by centrifugation at 28,000 x g for 10 min, suspended in 1.5 ml of 10 mM potassium phosphate buffer (pH 7.8) containing 5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol. The suspension was sonicated and centrifuged at 48,000 x g for 20 min. The supernatant solution (crude extract) was stored at -800C. Enzyme assays. The a subunit-specific reaction (a reaction) was assayed by a modification of the Smith and Yanofsky method (24). The reaction mixture (250 ,ul) included 0.18 M NaCl, 0.2 mM dithiothreitol, 0.05 mM pyridoxal phosphate, and 4 mM EDTA in addition to original constituents. When NH2OH was added, pyridoxal phosphate was omitted. After 20 min of incubation at 37°C, 50 ,ul of 1 M NaOH and 3 ml of water-saturated ethyl acetate were added, and the preparation was vigorously mixed. Aliquots (1.5 ml) of the cleared ethyl acetate layer were added to 2 ml of Ehrlich reagent, and the A540 was recorded. The 2subunit specific reaction (1 reaction) was assayed by the Kirschner et al. method (9); the overall a2132 subunit-specific reaction (ao3 reaction) was assayed by the Smith and Yanofsky method (24). Relative specific activities of wild-type and
1888
J. BACTERIOL.
LIM ET AL.
tt
tttt tt
t
tt
t
1.
ttt
1tt
7OKU14 LL2
LL
FIG.
t
Identity and distribution of amino acid substitutions in mutant
cx
subunits. The secondary structures for the S. typhimurium a
subunit for the mutagenized N-terminal, 121-residue region are shown along with turn (t) residues. Differences from the E. ccli enzyme are found at positions 6 (N-S), 12 (N-K), 13 (D-E), 15
(R->K),
42 (D-E), 52 (V-I), 68 (N-T), 90 (E-Q), 109 (N-K), 113 (A-*E), 117
(R-Q), and 120 (Q-K). Mutant designations are, for example, YC4, a Tyr-Cys change at position 4. Not listed above are the following mUltiPlY altered mUtant aL SUbUnitS: EGS/EG31, PS21/PL28, AV18/PL21, PT21/IV36, AT47/ATS9, GDS1/DN6O, PHS3/LQS8, GD75/AT86, TI77/YC102, AT74/FS114, GD75/DN112, TA77/HN92, QR8O/AV86, PS78IYC115, AT73/LP85, TA94/AV116, AV74/PS78, FS82/DN112, GD11O/MI1O1, LP85/AV103, FL72/GD98, LS11O/CR118, GS75/MI101, AT43/GSS1/ITS2, AV79/IV97/YC1O2, AT79/AT103/VA121, and
IV97/YC102/AV116.
mutant a subunits in crude extracts were measured in terms of pure 2 subunit with an excess of the a subunit. To insure that P2 (or a) subunit was added in excess, especially with crude extracts, a linear increase of activity with increased amounts of other subunit was monitored. Heat inactivation. The crude extracts (1 mg/ml) of at subunit in 0.1 M potassium phosphate (pH 7.8) containing 1 mM EDTA and 0.1 mM phenylmethylsufonyl fluoride were incubated at 52°C for 20 min. After treatment, they were cooled quickly on ice and assayed. The interval between heat treatment and enzymatic assay was kept constant for all proteins as a precaution to avoid the possibility that the activity of heat-treated a subunits may recover to some extent after heat treatment and during the assay. p-Reaction activity was utilized for several reasons. It is the most reliable among the three TSase reactions and circumvents major problems whereby some mutant a subunits exhibited varied levels of material in soluble form (12) and/or little or no detectable activities in the other TSase reactions. Calculation of relative amount of a subunit in crude extracts. The activity of each mutant a subunit was measured with various amounts of crude extracts in the ,B reaction in the presence of an excess of pure P2 subunit and calculated as units of au activity per milligram of pure 12 subunit. From the linear portion of this curve, the maximum activity can be determined, and comparison of these values with that of purified wild-type a subunit under similar conditions yields the relative maximum specific activity of the mutant a subunits. When the maximum specific activity of pure wildtype a subunit (units per milligram of a subunit) in the 1 reaction is known, the corresponding maximum specific activity of each mutant a subunit in this reaction can be estimated. Thus, the relative amounts of both wild-type and mutant a subunits (i.e., milligrams of a polypeptide per milligram of total soluble protein can be determined for any a subunit-containing crude extract. Solvent accessibility. The static solvent accessibility of residue in the X-ray crystal structure was calculated by the ACCESS program of Lee and Richards (10). Since only the structure of the a2P2 complex was known, the coordinates used for the a subunit were those remaining after the coordinates of 12 subunit, pyridoxal phosphate, and indole3-propanol phosphate were removed from the whole complex. The static accessibility of an amino-acid residue, X, in model tripeptides Ala-X-Ala as calculated in Lee and Richards (10) was used here as the reference accessibility of a fully extended polypeptide. When an amino acid had two
sets of data from different conformations, the average was adopted, and the solvent accessibility of Glu, Asp, and the a carbon of Gly was considered as those of Gln, Asn, and the side chain of Gly, respectively. Protein assays. The concentrations of purified a and 32 subunits were estimated from the extinction values (E278nm1% of 4.4 for the wild-type a subunit [1] and 6.5 for the 132 subunit [4]). The protein concentrations of crude extracts were measured by the microbiuret method (11). RESULTS AND DISCUSSION Altered residue sites in the mutant a subunits. The a subunit component of the a2132 TSase complex consists of alternating a helix and ,B-sheet structures in a a1-barrel motif (8). The trpA mutations identified in this study resulted in a subunits altered at residue sites within approximately the first half (residues 1 through 121) of the a polypeptide. Figure 1 shows the secondary structures within this region, the sites and identity of the single substitutions, and a list of multiple amino acid substitutions of mutant a subunits. A total of 64 residue sites have been altered in both ordered and random sequences. There are 49 different residue sites that have been singly altered with multiple amino acid substitutions at 16 of these sites. Thus, 66 mutant a subunits are now available containing single amino acid differences from the wild-type a subunit. As noted above, this region of the a polypeptide contains only two of the missense residue sites (Phe-22 and Glu-49) found in early random in vivo mutagenesis studies. All of the mutant trpA genes encoding these proteins are contained in overexpression vectors, which allow the expression of mutant a subunits'for study. As yet, none of mutant trpA genes encoding for the multiply altered a subunits has been subcloned into overexpression vectors. Instead we have focused our efforts here on describing initially the enzymatic and stability properties in crude extracts of the singly altered proteins. Enzymatic activities of mutant a subunits. The activity of each of the singly altered at subunits was assayed in crude extracts in the presence of a fixed amount of pure 132 subunit in the three TSase reactions (Table 1). Two broad classes of mutant a subunits can be seen; those (48 of 66) with normal activities and those (18 of 66) with altered activities in one or more of the reactions. Some general features of these results are worth considering at this point. First, as observed previously (18), about 25% of single-base-pair alterations lead to no amino acid
VOL. 173,
MUTANT E. COLI TRYPTOPHAN SYNTHASE at SUBUNITS
1991
1889
TABLE 1. In vitro and in vivo properties of mutant a subunitsa Mutants with defective activities
Relative sp act (%) in the following reaction:
Growth on minimal
EXPOSED 100
Defective in a and ot, reactions FS22
EG49 DN60 DG60 YC4 TK63 QR65 CR81 RL89
Defective in ,3 and ao reactions GD51 DG56
YC102 NS104 Defective in all TSase reactions PL28 PH53 PT53 AV67 CR118
REACTION
a
mediumb
44 0
