study of anthranilate synthase function by replacement of

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc

Vol. 260, No. 3, Issue of February 10, pp. 1889-1894,1985 Printed in U. S. A.

Study of Anthranilate Synthase Function by Replacement of Cysteine 84 Using Site-directed Mutagenesis*

(Received for publication, July 18, 1984)

Janet L. Paluh, Howard Zalkin, David Betsch, and H. Lee Weith From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

Cysteine 84 was replaced by glycine in Serratia marcescens anthranilate synthase Component I1 using site-directed mutagenesis of cloned trpG. This replacement abolished the glutamine-dependent anthranilate synthase activity but not the NH3-dependent activity of the enzyme. The mutation provides further evidence for the role of active site cysteine 84 in the glutamine amide transfer function of anthranilate synthase Com- ponent 11. By the criteria of circular dichroism, proteo- lytic inactivation, and feedback inhibition the mutant and wild type enzymes were structurally similar. The NH3-dependent anthranilate synthase activity of the mutant enzyme supported tryptophan synthesis in media containing a high concentration of ammonium ion.

Anthranilate synthase catalyzes the initial reaction in tryptophan biosynthesis in microorganisms and plants. This enzyme is one of a family of glutamine amidotransferases, enzymes that utilize the amide of glutamine in the biosynthesis of amino acids, nucleotides, coenzymes, and an amino sugar (1, 2). Glutamine amidotransferases thus exert a major role in utilization of assimilated nitrogen. Anthranilate synthase is the most thoroughly characterized glutamine amidotransferase. Its properties were instrumental in the formula- tion of a mechanism for glutamine amide transfer (3,4). The enzyme is an oligomer of nonidentical subunits designated AS I’ and AS 11. AS I catalyzes an NHS-dependent synthesis of anthranilate (Equation 1).

Chorismate + NHs + anthranilate + pyruvate (1)

AS I1 binds glutamine and provides glutamine amide transfer function to the AS I .AS I1 complex (Equation 2).

Chorismate + glutamine + anthranilate + pyruvate + glutamate (2)

Chemical modification experiments (5-8) have identified an AS I1 active site cysteine essential for glutamine amide transfer. As expected for an active site residue, this cysteine is conserved in 7 microbial AS I1 sequences (9-11).

In some enteric bacteria (9) and eukaryotic microorganisms (10, ll), AS I1 is covalently joined to other enzymes of tryptophan biosynthesis to yield multifunctional enzymes. In

* This work was supported by United States Public Health Service Grant GM 24658. This is Journal Paper 9960 from the Purdue University Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: AS I, anthranilate synthase Compo- nent I; AS 11, anthranilate synthase Component 11; bp, base pair(s); I, liter; u, unit(s); ECTEOLA, epichlorohydrin triethanolamine cellulose.

other bacteria, including Serratia marcescens (8, 9) AS I1 is monofunctional. In S. marcescens, trpE encodes AS I and trpG, AS 11. The native enzyme has an CY& quaternary structure in which the subunits are tightly associated (5). The S. marcescens AS I1 active site cysteine is residue 84 in the 193-amino acid primary translation product (8, 9).

In this paper we report the replacement of the S. marcescens AS I1 active site cysteine by site-directed mutagenesis of cloned trpG. We have examined the effects of a Cys-84 to Gly change on in vitro activity and in vivo function.

EXPERIMENTAL PROCEDURES

Strains, Plasmids, and Phage-Plasmid pGM6 contains an approximately 5-kilobase EcoRI fragment of S. marcescens DNA ligated into the EcoRI site of pBR322 (12). Plasmid pGM6 has intact trpEGDCB genes which are expressed and function in Escherichia coli. A Hind111 deletion derivative of pGM6, designated pSM61, lacking trpB function was obtained from Charles Yanofsky, Stanford University. Plas- mid pRK9, obtained from Rick Kelley, Stanford University, is an expression vector that utilizes the S. marcescens trp promoter. The M13 phage were M13mplO (lab stock) and M13mplOw (from Carl Bauer, University of Illinois). The designation “w” indicates that the phage does not contain amber mutations in genes I and 11. E. coli strains JM103, JM105, and GM119 for growth of M13 phage have been described (13). E. coli strain JMBS (thr, leu, thi, gal1,2, lac, xyl, ara, atrpLD102) was obtained from Charles Yanofsky, Stanford University, and was used as a recipient for plasmids carrying S. marcescens trp genes. The growth rates of strain JMBS transformants were measured in M9 media, pH 7.0 (14), 50 pg/ml each of threonine and leucine, 1 pg/ml thiamin, 50 pg/ml ampicillin, and NH&1 as specified.

Plasmid and Phage Constructions-A 3.4-kilobase EcoRIINruI fragment of S. marcescens DNA, trpE+G+D+, was isolated from plasmid pSM61 and was ligated into the EcoRI and Nrul sites of pBR322 to yield plasmid pJP l7 (Fig. 1). Plasmid pJP17 expressed trpEGD functions as determined by complementation of E. coli strain JMB9. For mutagenesis of trpG a 620-bp PuuII segment extending from nucleotide 58 in trpG to an unsequenced portion of trpD was cloned into the SmaI site of M13mplO. A recombinant was chosen which had the orientation EcoRI-ClaI-Bell-BamHI (Fig. 1). The resulting phage, designated M13mplOG620, contained a segment of the trpG coding strand from nucleotides 58-582 plus approximately 95 nucleotides of trpD.

Construction of Gapped Duplex-A gapped heteroduplex consisting of M13mplOG620 and M13mplOw was constructed according to a protocol supplied by Carl Bauer and Jeff Gardner, University of Illinois. This heteroduplex is shown in Fig. 2. M13mplOG620 was grown in strain GM119 (supE,dam) to yield virion DNA unmethylated at GATC dam recognition sites. M13mplOw was grown in strain JM105 for preparation of replicative form DNA which was methylated a t GATC sites. The Ml3mplOw DNA was digested with EcoRI and BamHI, and 10 pg of linearized double stranded DNA were mixed with 25 pg of M13mplOG620 (+)-strand DNA in 110 p1 of SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). The mixture was heated at 100 “C for 10 min, slow cooled to 65 “C, and then incubated at 65 “C for 12 h to form the gapped duplex. The mixture containing gapped duplex was precipitated with ethanol, dried, and stored frozen in 5 pl of HZO. It was assumed that this mixture contained approximately 2 pg of gapped duplex/pl which is equivalent to approximately 1 pg/pl

1889

1890 Site-directed Mutagenesis of Anthranilate Synthase

FIG. 1. Partial restriction maps of plasmids and phage. Top line, EcoRI/ NruI fragment from S. marcescens (solid line) ligated into pBR322 (dotted line). Numbers in parenthesis correspond to positions of trpG sequence. Squiggles represent boundaries between trp genes. The internal 620-bp PvuII fragment of pJP17 was ligated into the SmaI polylinker site of pUC13 to yield pUC13G620. The BamHI and EcoRI polylinker sites were used to transfer the fragment into M13mplO. The black dot in pJP2l represents the mutation of T- 250 to G. The maps are not drawn to scale.

Eco R I Barn H I

pUC13G620

M13mplOG620

CI E

W 8

I.+

2 z

c(

[L

W 8

Dam', wild type

Dam-, amber

';1 r" n

c(

0 0

[L

w " w " E Z Z Z -a' o = 2 w s

pJP21 1,:"- "" " """"

FIG. 2. M13mplOG620 gapped heteroduplex used for mutagenesis. The heteroduplex was constructed as described under "Experimental Procedures." The inner M13mplO (+)-strand contains an approximately 620-nucleotide PuuII segment of trpGD coding strand DNA. The open box represents the M13mplO polylinker. The outer (-)-strand is derived from M13mplOw cut at the EcoRI and BamHI polylinker sites. The mutagenic oligonucleotide with a one- base mismatch is shown by a short line with a black dot. Primer extension is indicated by dashed lines and arrows.

(0.4 pmol) of template (+)-strand M13mplOG620. M13 growth, cloning, and DNA isolation were essentially as described by Messing (15).

Primed Synthesis-A sample of 32P 5'-end labeled 17-mer (12 pmol) was annealed with 0.4 pmol of gapped duplex in 10 p1 of solution A as previously described (13). Primer extension and ligation were conducted as described (13). Samples were taken to verify incorporation of the 32P-labeled 17-mer and for transformation into E. coli strain JM105.

ferred from M13mplOG620m to plasmid pJP17 in 3 steps. First, the Reconstruction of trpG-The trpG Cys to Gly mutation was trans-

488-bp SalIIClaI trpC portion of pJP17 was ligated into the Sal1 and ClaI sites of pBR322. Next, the internal 401-bp BclI/ClaI trpG segment of the pBR322 recombinant was replaced by the comparable DNA from M13mplOG620m which contained the Cys to Gly change. Finally, the SalI/ClaI DNA in the pBR322 recombinant was used to replace the wild type SalIIClaI segment in pJPl7. The resulting plasmid containing S. marcescens trpEGD having the TGC to GGC

c(

[L

codon change was designated pJP19 (Fig. 1). The relevant genotype of pJP19 is trpGGID+. Plasmids pJP17 and pJP19 are identical except for the 1 trpG codon change. The mutant Gly-84 enzyme was designated anthranilate synthase (trpG1).

Oligonucleotide Synthesis-A 17-mer d(TCGGCATCGGCCTCG- GC) was synthesized by the solid phase phosphoramidite method (16). Purification was by high-performance liquid chromatography on a polyethyleneimine anion exchange matrix (17).

DNA Sequence Analysis-DNA sequences were determined by the procedure of Sanger et al. (18). The polyacrylamide/urea gel electrophoresis system described by Biggin et al. (19) was used for resolving the DNA fragments. A single T reaction was used to screen phage DNA for the T to G mutational change.

Enzyme Purification-In plasmids pJP17 and pJP19 trp operon gene expression is from a plasmid promoter. In order to increase gene expression the strong S. marcescens trp promoter lacking the atten- uator region was isolated from plasmid pRK9 and was ligated into the EcoRI site immediately upstream of trpE. Plasmids pJP17 (trpE+G+D+) and pJP19 (trpE+GID+) containing the trp promoter were designated pJP20 and pJP21, respectively. Anthranilate synthase was purified from strains JMBS/pJPZO and JMB9/pJP21. Cells were grown at 30 "C to late log phase in media containing salts (20), 0.5% glucose, 0.1% acid hydrolyzed casein, 1 mg/l thiamine, 40 mg/l each of threonine and leucine, 20 to 40 mg/l indole acrylic acid, and 50 mg/l ampicillin. The enzyme was purified by a modification of the previous procedure (5). The changes were cell disruption in a French pressure cell instead of by sonic disruption, use of DEAE-Sepharose instead of DEAE-cellulose, and a final gel filtration on tandem 2.5 X 80-cm Sephacryl S300 columns replaced ECTEOLA-cellulose and hydroxylapatite column chromatographies. The enzyme was greater than 95% pure according to sodium dodecyl sulfate acrylamide gel electrophoresis (21). The yield was approximately 185 mg of enzyme/ 30 g of cells, wet weight. Enzyme was stored a t -20 "C in buffer solution containing 0.05 M KPO,, pH 7.0, 0.1 mM EDTA, 0.2 mM dithiothreitol.

Enzyme Assay-Glutamine and NHs-dependent anthranilate synthase were assayed a t 23 "C as described (5).

Enzyme Characterization-Circular dichroism spectra were recorded with a Cary 60 spectropolarimeter equipped with a Cary 6002 circular dichroism attachment as described by Brunden et al. (22). Enzyme samples were prepared in buffer solution containing 0.05 M potassium phosphate, pH 7.0, 0.1 mM EDTA, 0.2 mM dithiothreitol.

Site-directed Mutagenesis of Anthranilate Synthase 1891

The protein content of each sample was determined by the method of Lowry et al. (23). The secondary structure content of the enzymes was analyzed by the revised procedure of Provencher and Glockner (24), employing data points at 1-nm intervals.

Tryptophan inhibition of NHs-dependent anthranilate synthase was determined as previously described (5). Tryptic digestion as a probe of conformation (25) was conducted at 23 "C in 0.05 M triethanolamine, pH 8.9, with 1.67 mg/ml trypsin. Samples of 20 pl were removed for enzyme assay into a 1.0-ml reaction mixture. Phenyl- methanesulfonyl fluoride (0.75 mM) was added to inhibit proteolysis and the enzyme assay was completed within 1.5 min.

RESULTS

Mutant Isolation-A TGC (Cys) to GGC (Gly) codon change in S. marcescens trpG was constructed by incorporation of a synthetic oligonucleotide into a recombinant M13 heteroduplex. The nucleotide and amino acid sequence of the pertinent region of trpG together with the sequence of the 17- mer are shown in Fig. 3. The synthetic 17-mer has a single base mismatch at trpC nucleotide 250. The mutagenic oligonucleotide was annealed to the complementary region of the trpG coding strand of a gapped heteroduplex as described under "Experimental Procedures." The essential features of the heteroduplex, designated M13mplOG620, are noted in Fig. 2. The inner (+)-strand contains (i) an approximately 620-nucleotide portion of trpGD (Fig. 1) ligated into the M13mplO polylinker region, (ii) amber mutations in two essential phage genes, and (iii) unmethylated GATC dam methylation sites. The outer (-)-strand of the heteroduplex was derived from wild type M13mplO and was grown in a dam+ host to yield GATC-methylated DNA. After annealing the 17-mer, gaps were filled in with DNA polymerase Klenow fragment, and the mixture was treated with T4 DNA ligase.

Control reactions verified that the mutagenic oligonucleotide annealed to the correct site and was incorporated into the gapped duplex. First, the mutagenic oligonucleotide served as a dideoxy sequencing primer with M13mplOG620 template DNA and yielded the correct nucleotide sequence. Second,'a portion of the reaction mixture from the mutagenesis employing 5'-32P-labeled 17-mer was digested with EcoRI plus BamHI. After electrophoresis and autoradiography, a 32P- labeled band corresponding to the excised trpG fragment was detected.

A portion of the reaction mixture containing approximately 1 pmol of duplex DNA after primer extension and ligation was transformed into E. coli strain JM105 and yielded 70 clear plaques. DNA from 36 plaques was screened using the dideoxy T sequencing reaction. Fig. 4 shows a portion of the sequencing gel from which one mutant, in lane 4, having a T to G change was identified. This mutant was designated M13mplOG620m. Sequencing of the PuuII 620 fragment from trpG nucleotides 77-315 in M13mplOG620m verified that the mutant differed from the wild type only by a T to G change at nucleotide 250. A portion of the sequencing gel spanning nucleotides 149-315 is shown in Fig. 5.

To reconstruct intact mutant trpEG a portion of trpG,

24 1 250 258 I 1 I

trpG ATC GGC ATC TGC CTC GGC Ile Gly Ile CYS Leu Gly

171ner 5' -TC GGC ATC GGC CTC GGC-3 ' FIG. 3. Nucleotide sequence of a pertinent region of trpG

and of a synthetic 17-mer used for mutagenesis. The wild type sequence is shown on top. The nucleotide sequence of trpG is num- bered from the start of translation. The synthetic 17-mer having a one-base mismatch is shown below. The mismatch is underlined.

-

1 2 3 4 5 6

Dm

FIG. 4. Screening for a mutant having a T to G base change. The photograph shows a portion of the T reaction sequencing gel with 6 DNA samples. The arrow points to the pertinent T. DNA in lane 4 is from the mutant having the T to G change.

nucleotides 116-516, was excised from M13mplOG620m replicative form using BclI and ClaI (Fig. 1) and used to replace the comparable region in plasmid pJPl7 as described under "Experimental Procedures." The resulting plasmid, pJP19, is isogenic with pJP17 (trpE+G+D+) except for the Cys to Gly replacement at residue 84 in the trpC protein. The Cys to Gly mutation was designated trpG1.

Enzyme Activity-Anthranilate synthase activity was determined in extracts of plasmid-bearing cells. Glutamine- and NH3-dependent anthranilate synthase activity was 7.4 and 3.6 u/mg, respectively, from pJPl7 (trpE+trpG+trpD+). Glu- tamine-dependent anthranilate synthase was undetectable (<0.002 u/mg) in extracts from cells bearing pJP19 (trpE+trpGl trpD+) whereas the NH3-dependent activity was 7.1 u/mg. Following purification of the mutant enzyme to homogeneity, NH3-dependent anthranilate synthase was 2160 u/mg, whereas the glutamine-dependent activity was less than 0.01 u/mg. By comparison, specific activities of 3200 u/mg and 2150 u/mg for the glutamine- and NH3-dependent activities, respectively, were determined for wild type enzyme purified from plasmid bearing cells.

Comparison of Mutant and Wild Type Anthranilate Syn- the-Anthranilate synthase (trpC1) was compared to the wild type enzyme to determine whether conformational dif- ferences could be detected. The circular dichroism spectrum of the wild type enzyme is shown in Fig. 6. The spectrum of anthranilate synthase (trpG1) was indistinguishable. The secondary structure content, calculated by the method of Prov- encher and Glockner (24), is tabulated in Table I. The con- tents of a-helix and &structure are similar although differ-


G A C T " -

""

Ilr "

rc ZI

4

'c " - ab-

G A C T

w 3 .-A

4

4

\[- A

C 5l

FIG. 5. Nucleotide sequence of wild type (left) and trpGZ (right). A partial sequence from trpC nucleotides 149-315 is shown surrounding the annealing site of the mutagenic oligonucleotide. The arrows encompass the region of the sequence corresponding to the 17-mer. The TGC (Cys) and GGC (Gly) codons are boxed. The T to G change is marked by dots.

4 2 1 , , , , 205 215 225 2 35

Wavelength (nm)

FIG. 6. Circular dichroism spectrum of wild type anthranilate synthase. The spectrum was recorded as described under "Ex- perimental Procedures."

ences of 2-8% in 0-helix and P-turn would not be detected. Feedback inhibition of NH3-dependent anthranilate syn-

thase was identical for both enzymes. Approximately 7 PM

TABLE I Secondary structure calculation for wild type and mutant

anthranilate synthase Secondary structure was calculated as described under "Experi-

mental Procedures." Each value is a mean f standard deviation for three spectra on two enzyme samples.

Enzyme a-Helix &Structure Remainder 96

Wild type 30 f 2 25 f 6 46 f 4 Mutant 29 f 3 24 f 8 47* 7

300

200

IO0

8 50 c .I- - Q)

Y v

I- I-

zo [L c3

IO

5

I I I

L -I 2 6 IO 14 18

TIME (hours) FIG. 7. Growth of E. coli JMBS (AtrpEGD) transformants

carrying pJP17 (trpE+G+D+) or pJP19 (trpE+GZD+). Curve I , pJP17,50 mM NH4CI; curue 2, pJP17, 1 mM NH4Cl; curue 3, pJP19, 50 mM NH,Cl; curue 4, pJP19, 1 mM NH4CI; curue 5, pJP19, 1 mM NH4Cl plus 50 pg/ml tryptophan.

tryptophan was required for 50% inhibition. Loss of NHS- dependent anthranilate synthase activity resulting from pro- teolytic digestion by trypsin was also used for qualitative comparison of enzyme conformation. Inactivation by trypsin exhibited apparent first order kinetics. The half-time for inactivation was 13 min for both enzymes.

In Vivo Function-The in vivo function of S. mrcescens anthranilate synthase was evaluated in E. coli. Plasmid pJPl7 (trpE'G'D') complemented E. coli strain JMBS (AtrpEGD) and allowed growth at the wild type rate indicating that the Serratia enzyme was completely functional in E. coli. The data in Fig. 7 show that JMB9/pJP17 grew with a doubling time of 1.1 h in minimal media containing 50 mM NH&l as nitrogen source. Addition of tryptophan did not change this growth rate. In medium with low NH4Cl (1 mM) the growth rate of this strain was somewhat reduced with a doubling time of 1.4 h. The data in Fig. 7 show that plasmid pJP19 (trpE+trpGltrpD+) conferred a Trp' phenotype in media with 50 mM NH4Cl (doubling time 2.8 h) but that growth was


severely restricted in media with low NH4C1 (doubling time, 14 h). Addition of tryptophan restored the wild type growth rate to strain JMB9/pJP19 in media with low NH4C1. These results establish that NH3-dependent anthranilate synthase can function in tryptophan synthesis when the concentration of NH4C1 in the medium is high.

DISCUSSION

In plasmid pJP17 S. marcescem genes trpEGD are tran- scribed from a plasmid promoter and are translated to yield anthranilate synthase (trpEG) and anthranilate phosphori- bosyltransferase ( t rpD) , both of which are functional in E. coli. By site-directed mutagenesis trpG codon 84 TGC (Cys) was changed to GGC (Gly).

We have utilized a mutagenesis protocol that requires annealing a synthetic oligonucleotide to an M13-derived gapped heteroduplex (13). This heteroduplex was designed to enrich for mutant progeny by taking advantage of the E. coli dam methylation system and an amber selection. Dam methylation is possibly important because in cases of asymmetrical methylation of heteroduplex molecules there is a bias in marker recovery that favors the marker encoded by the methylated strand (26). The asymmetric methylation of the gapped heteroduplex (Fig. 2) may in part compensate for 7 nonmethy- lated GATC sites in the M13mplOG620 trpG DNA that result from in vitro primer extension. The desired T to G mutation a t trpG nucleotide 250 in the (-)-strand is preceded by non- methylated GATC sites at nucleotides 102, 117, and 237 and followed by sites at 267, 309, 333, and 427. I t is not known how well methylation of the 8 viral GATC sites in the M13mplO (-)-strand compensates for the unfavorable methylation pattern in the cloned trpG insert. The amber selection likewise favors recovery of the mutation from the (-)-strand of the heteroduplex. After transformation of the heteroduplex into the JM105 sup+ host, the M13mplOG620 (+)-strand which is trpG+ cannot replicate because of amber mutations in essential M13 genes. We have not undertaken to optimize the mutagenesis which yielded one mutant in 36 phage clones screened.

Three main conclusions emerge from analyses of the trpG Cys-84 to Gly replacement: (i) Cys-84 has an essential role in glutamine amide transfer; (ii) structural or conformational alterations in anthranilate synthase, if they exist, were not detected as a consequence of the Cys-84 to Gly replacement; (iii) NH3-dependent anthranilate formation can support tryptophan synthesis when the concentration of NH&l in the growth medium is sufficiently high. Previous evidence for the role of AS I1 Cys-84 in glutamine amide transfer was obtained from specific inactivation of glutamine-dependent anthranilate synthase by affinity labeling and group specific modification (3, 5-8). As a result of the AS I1 Cys-84 to Gly replacement we have confirmed the essential function of Cys-84 in glutamine amide transfer. Replacing Cys-84 with Gly elimi- nates the possibility that chemical modification of Cys-84 blocked glutamine amide transfer because of introduction of bulky modifying reagents rather than by specific chemical modification of an essential cysteine.

However, even in the case of amino acid replacement, alternative conclusions are difficult to exclude. For example, amino acid replacement might cause a relatively minor structural alteration that could abolish glutamine binding or amide transfer independent of the function of Cys-84. Although it is impossible to unequivocally exclude the possibility of changes in secondary structure short of an x-ray crystallographic determination, we could not find evidence for structural dif- ferences in native and mutant anthranilate synthase by cir-

cular dichroism, proteolysis by trypsin, or feedback inhibition by tryptophan.

The data in Fig. 7 support the conclusion that NH3-dependent anthranilate synthase was functional for tryptophan synthesis. The function of NH3-dependent anthranilate synthase in tryptophan synthesis does not result from elevated enzyme levels due to a multicopy plasmid. Elevated enzyme levels were not obtained because trp operon gene expression from plasmids pJP17 (trpE+G+D+) and pJP19 (trpE+GID+) was dependent upon a weak plasmid promoter. In previous experiments (27, 28) trpG deletion strains were used to dem- onstrate that AS I could function in tryptophan synthesis. The growth curves in Fig. 7 show that the biosynthetic capac- ity of NH3-dependent anthranilate synthase from plasmid pJP19 was dependent upon the NH4C1 concentration in the medium. The dependence of growth rate on NH&l concentration reflects the high K,,, for NH3 ( 5 ) .

We have reported that the NH3-dependent activity of mu- tationally altered glutamine phosphoribosylpyrophosphate amidotransferase can function in purine nucleotide synthesis under similar conditions of high NH4C1 level (13). If primitive glutamine amidotransferases were strictly NH3 dependent (3), there would be strong selective pressure to improve the effi- ciency of biosynthesis by recruiting a glutamine amide transfer function. An ancestral gene having glutamine amide transfer function could have supplied a trpG AS I1 subunit to trpE- encoded AS I. We have recently obtained evidence for a trpG- related glutamine amide transfer domain in E. coli GMP synthetase (29). Thus a glutamine amide transfer domain in anthranilate synthase, p-aminobenzoic acid synthase (30), and GMP synthetase has evolved from a common ancestor.

Acknowledgments-This project was initiated in the laboratory of Charles Yanofsky, Stanford University, and benefited accordingly. We are grateful to Carl Bauer and Jeff Gardner, University of Illinois, for communicating their mutagenesis protocol prior to publication and to Kurt Brunden and William Cramer, Purdue University, for obtaining circular dichroism spectra and assisting in data analysis.

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study of anthranilate synthase function by replacement of

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