Expression of glr (mud, dga) - Journal of Biological Chemistry

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 32, Issue of November 15,pp. 24242-24246, 1993 Printed in U.S A .

Expression of glr ( m u d , dga)Gene Encoding Glutamate Racemase in Escherichia coli* (Received for publication, April 27, 1993)

Tohru Yoshimura, MakotoAshiuchi, Nobuyoshi Esaki, Choei Kobatake, Soon-Young Choi, and KenjiSoda3 From the Institute forChemical Research, Kyoto University, Uji,Kyoto-Fu 611, Japan The m u d ( d g agene ) of Escherichia coli is required for the biosynthesis of D-glutamate, an essential cornponentof bacterial peptidoglycan (Doublet, P., van Heijetnoort, J., and Mengin-Lecreulx, D. (1992)J. Bacteriol. 174, 5772-5779; Dougherty, T. J., Thanassi, J. A., and Pucci, M. J. (1993)J . Bacteriol. 175, 11 1-1 16),but its geneproduct has not been identified. We found that the amino acid sequence of protein deduced from the nucleotide sequence of the open reading frame of murl gene (ORF 1)shows a significant homology with that of glutamate racemase of Pediococcus pentosaceus. The amino acid sequence of glutamate racemase of Lactobacillus fermenti recently reported also shows a homology with the deduced amino acid sequence of ORFI (Gallo, K. A., and Knowles, J. R. (1993)Biochemistry 32,3981-3990).The mur1 (dga) gene was ligated into a plasmid, pKK223-3, with a designed ribosome binding site and expressed in E. coli JM109 cells. Glutamate racemase was produced bythe transformant cells, whereas theenzyme was not found in the host cells. Accordingly, we newly termed the gene glr, which is more relevant than murI and dga. We partially purified the enzyme to characterize it. The enzyme consists of two identical subunits with a molecular weight of about 31,000in contrast to the P. pentoeaceus enzyme, a monomerprotein.

Bacterial cell walls contain several kinds of D-amino acids as components of peptidoglycans, and these D-amino acid residues are thought to protect the cell walls from protease action. D-Glutamate is incorporated into peptidoglycan through its addition to UDP-N-acetylmuramyl-L-alanine, a peptidoglycan precursor, and this reaction is catalyzed by UDP-N-acetylmuramoylalanine-D-glutamate ligase (EC 6.3.2.9) (1).D-Glutamate can be synthesized from D-alanine and a-ketoglutarate by D-amino acid aminotransferase (EC 2.6.1.21) (2-6) and alternatively from L-glutamate by glutamate racemase (EC 5.1.1.3) (7-11). However, both enzymes are found only in some bacteria. D-Amino acid aminotransferase in bacilli and Rhodospirillum rubrum (12), glutamate racemase in lactobacilli and Pediococcuspentosaceus, and their physiological functions have not been fully elucidated. It is unknown if their roles are biosynthetic, biodegradative,

* 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 nucleotide sequencefs)reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank withaccession numberfs) L22448. $ To whom all correspondence should be addressed. Tel.: 81-77433-2594; Fax: 81-774-33-1271.

or both. The biosynthetic pathway of D-glutamate in other bacteria such as Escherichia coli has not been shown. Recently, the gene responsible for the D-glutamate biosynthesis in E. coli was identified and designated as m u d (13) and later as dga (14). We here tentatively use the term of murl, but newly termed it glr as described later. The gene complements the D-glutamate auxotrophy of the E. coli strain, WM335. Sequencing of the gene has revealed that thegene corresponds to a previously sequenced open reading frame, ORFI (15). The m u r l gene product was predicted to consist of 289 amino acid residues with a molecular weight of 31,504 (15) and contain a large number of hydrophobic amino acid residues. However, attempts were made to identify the enzyme activity of the gene product without success (13). We have been studying the structure and function of glutamate racemase of P. pentosaceus (10,11, 16, 17). Recently, we cloned and sequenced its gene' and found that the amino acid sequence of glutamate racemase shows a significant homology with that of the m u d gene product. Gallo and Knowles (18) also found that the amino acid sequence of glutamate racemase of Lactobacillusfermenti showed 30% homology with that of the m u d gene product. We expressed the m u r l gene and demonstrated glutamate racemase in the transformant cells. Wenow provide the evidence that the m u d gene is expressed in the cell of E. coli JM109 to produce glutamate racemase, which is involved in the synthesis of Dglutamate. We also show that D-glutamate is a direct precursor of the glutamyl residue of peptidoglycan in E. coli. EXPERIMENTALPROCEDURES

Materials-All restriction enzymes and the T4 DNA ligation kit were purchased from Takara Shuzo (Kyoto, Japan). TaqDNA polymerase was from Promega Corp.; plasmid pKK223-3 was from Pharmacia LKB Biotechnology Inc.; all reagents for DNA synthesis was from Applied Biosystems, Inc.; the protein assay reagent and the SDS-PAGE' standard was from Bio-Rad; standard proteins for gel filtration were from Oriental Yeast (Japan); l-fluoro-2,4-dinitrophenyl-5-l-alanineamide (FDAA, Marfey'sReagent) was from Pierce Chemical Co.; L-glutamate oxidase was a kind gift from Dr. H. Kusakabe of Yamasa Shoyu (Japan). L-[U-"C]Glutamate (9.25 GBq/ mmol), ~-[U-"C]proline (9.25 GBq/mmol), and [U-''C]a-ketoglutarate (7.4 GBq/mmol) were obtained from Du Pont-New England Nuclear. All other chemicals were of analytical grade. The Koharaordered X phage bank, XEllCll (19), was a kind gift from Dr. y. Kohara of the National Institute of Genetics, Japan. Construction of Plasmids pGR2 and pGR3"hEllCll phage DNA was digested with EcoRI and HindIII. The plasmid pGR2 was obtained by insertion of the resultant 1.9-kilobase pair fragment containing the murl gene into pKK223-3 (Fig. 2). Plasmid pGR3 contained the ORF of the murI gene (ORFI) with a designed ribosome

* S.-Y. Choi, N. Esaki, M. Ashiuchi, T. Yoshimura, and K. Soda, manuscript in preparation. The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame.

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E. coli Glutamate Racemase binding sequence. This was constructed by means of the polymerase chain reaction method with nrimers, GRP-1 and GPR-2, which were synthesized on an Anplied Biosystems 381A DNA synthesizer. -two primers were 5’-AGCCAGAATTCSequences of the AGGACAAAGACCATGAGAC-3’ (GRP-1. N terminus) and 5’CAACATTCCTGCAGATCAGCCTAAA-3” (GRP-2, C terminus). GRP-1 contained an EcoRI restriction site (underlined) and a designed ribosome binding sequence (boldface type). The reaction mixture for the polymerase chain reaction (100 ~1) consisted of 8 pmol of Tris-Cl buffer (pH 8.3), 2 pmol of (NH&SO,, 0.5 pmol of MgC12, 20 pmol of each NTP, 2.5 units of TaqDNA polymerase, 1 pg of XEllCll phage DNA (as a template), and 100 pmol of each GRP-1 and -2. The reaction mixture was heated at 94 “C for 1 min (for denaturation), then cooled ranidlv to 52 “C with a 1-min hold (for annealing), and incubated at 72 “C for 4 min (for extension). The programed temperature shift was repeated 40 times. The plasmid pGR3 was constructed by replacement of the 38base pair EcoRI-PstI fragment of PGR2 by the corresponding fragment excised from the polymerase chain reaction product. Enzyme and Protein Assay-Glutamate racemase was assayed with D-glutamate as a substrate by determination of a-ketoglutarate formed from L-glutamate, the product of racemase reaction with Lglutamate oxidase. The reaction mixture (200 ~1) containing 20 pmol of Tris-Cl buffer (pH 8.0), 2 pmol of D-glutamate, and enzyme was incubated at 30 “C for 1 h. The reaction was terminated by addition of 10 ~1 of 12 M HCl. After the reaction mixture was neutralized by addition of 20 rd of 6 M NaOH, a 160-~1 aliquot was withdrawn from the mixture and incubated at 37 “C for 1 h with 20 rrmol of Tris-Cl buffer (pH 8.0) and 0.5 unit of L-glutamate oxidase in a final volume of 200 pl. The reaction mixture was mixed with 200 ~1 of 0.066% dinitrophenylhydrazine in 2 M HCl, then stood at room temperature for 10 min. and mixed with 1.0 ml of 2 M NaOH. a-Ketoglutarate that formed was determined by measurement of an increase in the absorbance at 550 nm. One unit of enzyme was defined as the amount that catalyzes the formation of 1 nmol of product/min. Glutamate racemase was alternatively assayed with high performance liquid chromatography by determination of L-glutamate formed from D-glutamate after the product was derivatized to a diastereomer with Marfey’s reagent (20). The reaction mixture (1 ml) containing 20 pmol of Tris-Cl buffer (pH 8.0), 10 pmol of D-glutamate, and enzyme was incubated at 30 “C for 2 h, and the reaction was terminated by addition of 50 pl of 12 M HCl. After the reaction mixture was neutralized with 100 ~1 of 6 N NaOH, a loo-p1 aliquot was withdrawn and mixed with 200 ~1 of 1% Marfey’s reagent in acetone and 40 rl of 1 M NaHC03 (21). The reaction mixture was incubated at 37 “C for 1 h in the dark,.and the reaction was stopped by the addition of 20 al of 1 M HCl. The reaction mixture was dried up with a Speed Vat Concentrator (Savant), and the residue was dissolved in 1.4.ml of methanol. FDAA-DL-glutamate thus obtained was applied to a TSK ODS-12OT column (0.46 X 25 cm Tosoh. Jaoan) eauiDoed on a Waters 600 high performance liquid chromatography system. The column was developed at a flow rate of 0.6 ml/min with a linear gradient of acetonitrile concentrations (10-40%/h) in 50 mM triethylamine-phosphate buffer (pH 2.8) with monitoring the absorbance at 340 nm. Protein concentration was determined with a Bio-Rad protein assay kit with bovine serum albumin as a standard. -

I

I

Expression and Purification

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.

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.

of Glutamate Racemase Encoded by the

murl Gene-E. coli JM109 cells were transformed with DGR~ or pGR3. Each transformant was cultured overnight in 5 ml of the Luria-Bertani’s broth supplemented with ampicillin (50 rg/ml). The culture was then inoculated into the fresh medium (500 ml) containing 0.1 mM isopropyl+D-thiogalactopyranoside. The cells were harvested after incubation at 37 “C for 10 h. The cells (1.4 e of wet weight) were suspended in 5 ml of the standard buffer con&ting of 50 mM Tris-Cl (pH 7.5), 1 mM DL-glutamate, 10% glycerol, 0.1% 2mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride and disrupted by sonication. After centrifugation, the supernatant solution was dialyzed against the same buffer and used as a cell extract. The precipitate (pellet) was resuspended in the standard buffer supplemented with 6 M urea and incubated at 4 “C for 16 h. After centrifugation, the supernatant solution was dialyzed against the standard buffer. The resultant solution was designated as a cell pellet extract. The cell extract for purification (3680 mg of protein and 17,000 units of glutamate racemase) was prepared from 34 g (wet weight) of E. coli JM109/pGR3 cells and applied to a DEAE-cellulose column (4.8 X 25 cm) equilibrated with the standard buffer. The column was

washed with the standard buffer and subsequently with the same buffer containing 0.2 M NaCl. The enzyme was eluted with 1200 ml of a linear zradient of NaCl concentrations (0.2-0.5 M) in the standard buffer. The active fractions (1100 mg of protein, 12;800 units) were combined, concentrated, and subjected to gel filtration with a HiLoad 26/60 column (Pharmacia) equipped on a fast protein liquid chromatography system (Pharmacia). The column was equilibrated with the standard buffer containing 0.5 M NaCl and developed with the same buffer at a flow rate of 3.5 ml/min. The active fractions (24.8 mg, 467 units) were combined and used as a partially purified enzyme. Molecular Weight Determination of Glutamate Racemase-Molecular weight of glutamate racemase was determined by gel filtration with a G3000sw column (0.75 X 30 cm. Tosoh, Japan) equilibrated with 50 mM Tris-H,SO, buffer (pH 7.5) containing 0.2 M Na&04, 1 mM DL-glutamate, 10% glycerol, 0.1% 2-mercaptoethanol at a flow rate of 0.1 ml/min. A calibration curve was made with the following proteins: alcohol dehydrogenase (Mr = 150,000), bovine serum albumin (66,200), carbonic anhydrase (29,000), and cytochrome c (12,400). Preparation of D-[U-‘4C]GZutamate-D-[U-14C]Glutamate was synthesized from [U-i4C]cu-ketoglutarate and D-alanine with D-amino acid aminotransferase (22). The reaction mixture (10 ~1) containing 100 mM potassium phosphate buffer (pH 8.0), 18 mM [U-‘4C]aketoglutarate (1.85 MBq), 20 mM D-alanine, 20 mM NADH, 1 FM pyridoxal 5’-phosphate, 0.12 unit of D-amino acid aminotransferase, and 0.1 unit of lactate dehvdroaenase was incubated at 50 “C for 30 min. The reaction mixture was-mixed with 0.1 ml of 1 M HCl and applied to a Dowex 50-X8 column (H’ form, 0.5 ml). After the column was washed with water, D-[U-‘4C]glutamate (total radioactivity, 0.892 MBq) was eluted with 1 M NH,OH. Incorporation of the ‘%-Labeled D-Glutamate, L-Glutamate, L-Proline, and a-Ketoglutarate into the Peptidoglycan of E. coli K12 CellsE. coli K12 cells were cultured in 5 ml of M9 minimal medium (23) containing 2% glucose at 37 “C for 20 h. This culture was then inoculated into a fresh medium (100 ml) containing each 740 kBq of L-[U-14C]glutamate (about 80 nmol), D-[U-r4C]glutamate (72 nmbl), L-IU-“Cloroline (80 nmol). or lU-14Clor-ketozlutarate (100 nmol) at 37’“C for-20 h. The cells harvested (wet weight, 0.3 g)‘were washed with 0.9% NaCl and mixed with 1.7 g (wet weight) of E. coli K-12 cells cultured with M9 minimal medium supplemented with 2% glucose. Peptidoglycan was purified as follows. The cells suspended in 10 ml of H,O were mixed with 50 ml of 4% SDS and boiled for 60 min. After being incubated at room temperature with stirring for 3 h, the mixture was centrifuged by Beckman L7-55 ultracentrifuge and SW 28 rotor at 25,000 rpm at 25 “C for 80 min. The precipitate was dissolved in an appropriate volume of hot water and centrifuged again under the same conditions. The precipitate was again dissolved in a small volume of water, mixed with 50 ml of 4% SDS, and boiled for 30 min. The mixture was incubated at room temperature with stirring and centrifuged under the same conditions. The precipitate was dissolved in hot water and centrifuged again. This washing step was repeated seven times, then the pellet was dissolved in 50 ml of 10 mM Tris-Cl buffer (pH 7.4), and digested with Pronase E (10 mg) at 37°C overnight. The reaction mixture was mixed with 2 g of SDS and boiled for 30 min, followed by incubation at room temperature for 3 h. The mixture was centrifuged under the same conditions as above and washed once. The precipitate obtained was dissolved in hot water and centrifuged with an SW 55 Ti rotor at 45,000 rpm for 60 min. This step was repeated seven times. The precipitate obtained was lyophilized and used as a purified peptidoglycan. The radioactivity of D-glutamate in the peptidoglycan was determined as follows. The purified peptidoglycan (0.5 g) was hydrolyzed at 108°C for 25 h in 6 N HCl containing 1% (v/v) phenol with a Waters Pica-Tag Work Station. The hydrolysate was subjected to amino acid analysis with a Hitachi-835 amino acid analyzer. The eluate from the analyzer was fractionated, and radioactivity of the fraction containing glutamate was determined with a Packard Tri-Carb scintillation spectrometer with Clear-sol I (Nacalai Tesque, Japan) as a scintillator. RESULTS

AND

DISCUSSION

Sequence Homology of murl Gene-encoding Protein of E. coli with Glutamate Racemases of P. pentosaceus and L. fermenti-Linear alignment of the amino acid sequence of the murl gene-encoding protein of E. coli and those of the glutamate racemases of P. pentosaceus’ and L. fermenti (18) are shown in Fig. 1. The overall sequence homologies between the ORFl and the glutamate racemases are about 30% and are

E. coli Glutamate Racemase

24244

MRQSMATKLQDGNTPCLAATPSEPRPTVLV -E. coli (1-40) MDNRPIGFP. pentosaceus (1-18) L. fermenti (1-18) MDNRPIGV-~

FDSGVGGLSV MDSGVGGLTV MDSGVGGLSV

YDEIRHLLPD LHYIYAFDNV AFPYGEKSEA VKTAQKUPN EEIIFIGDEA RMPYGPRPTA VRVIWKLPN EEVIFVGIQG HFPYGTKDQA

FIVERVVAIV EVVEFSRQMA EVRQLALSIG

(41-80) (19-58) (19-58) (81-120) (59-97) (59-97)

TAVQERYPLA LAVVACNTAS TVSLPALREK FDFPVVGVVP SFLNTKNIKA LVIACNTAT NAALAVLQAE LPIPVIGVIL AFLLKHDVKM M-VVACNTAT AAALPALQAA LPIPVIGVIE

(121-159) (98-136) (98-136)

AIKPAA-RLT ANGIVGLLAT RGTVKRSYTH ELIARFANEC PGAIAANRQT KNQKIGVIAT LGTIKSEAYP KALAEINTKL PGARAALAQD KKGPIGVIAT TATlTAGAYP ATIERLAPGT

# PCR

EcoRVHindlll

/

/ I . .+:.:, I \ EcoRI

PSI

I

I I

I

Metnsp

(160-199) (137-176) (137-176)

QIEMLGSAEM VELAEAKLHG EDVSLDALKR ILRPWLRMKE RAYPVACQEF VEIAEKNELH ITAAQKVMNE KLA-EFRQDQ PVIAKATQPM VEIVEHCQTG TAKAQEVVSE QLM-TFKEHP

(200-238) (177-215) (177-215)

PPDTVVLGCT HFPLLQEELL QVLPEGTR-L VDSGAAIARR -1DTLILGCT KFPLL-EEGI QAAVGPDVTL VDPGVETVHQ -VKTLIMGCT HFPLAPEI SKAVGPTVAL VDPAKETVAT

(239-278) (216-255) (216-254)

TAWLLEHEAP DAKSADANIA FCMAMTPGAE QLLPVLQRYG LIEILTKQAL QHAEGPKAQD QYYSTCNIKN FEEIARTFLN AKSWLEQHQA MGNHAHPNYH L-YSTCNLPD LRACVNKWLL

(279-289) (256-265) (255-268)

FETLEKLAVL G QDLRVEEVKI D SCHFDLGTAQ IEEGD

FIG.1. Linear alignment of the amino acid sequence the of predicted murI gene product of E. coli (upper), glutamate and L. fermenti (lower). racemaseof P.pentosaceus (middle), Three sequences were aligned by introducing gaps (hyphens) to maximize identities. Common residues in two or three enzymes are shown by boldface type. lower than thatbetween the two glutamate racemases (44.5%, 118 out of 265 matchable residues). However, the homologous local sequence of three segments (31-39,93-100, and 206-214 of the ORFlsequence) is highly conserved in three enzymes. Both glutamate racemase reactions are considered to proceed through a two-base mechanism (17, 24). Conversion of Cys73 and Cys-184 of glutamate racemases of the L.fermenti (24) and P.pentosaceus3 to alanyl residues by site-directed mutagenesis resulted in a loss of the activity. Therefore, these cysteinyl residues are thought to serve as a catalytic base in both glutamate racemases; the bases probably remove the ahydrogen from the substrate glutamate to result in racemization. These cysteinyl residues are found in the above homologous segments, and thecorresponding cysteinyl residues are conserved in the mud gene-encoding protein as cys-95 and Cys-209. These strongly suggest that the murI geneencoding protein is identical with glutamate racemase. Expression and Identification of the mud Gene-We constructed the plasmid pGR2 and transformed E. coli JM109 with it to obtain the m u d gene-encoding protein (Fig. 2). However, no glutamate racemase activity was detected in the cell extract of E. coli JM109/pGR2 cells. SDS-PAGE of the cell extract showed the absence of the protein with a molecular weight of about 31,000; the molecular weight of the murI gene-encoding protein was calculated as 31,504 based on the sequence (data not shown). We supposed that failure of the murI gene to be expressed was due to itsunsuitable structure, in particular the lack of the definite ribosome binding sequence upstream of the ATG initiation codon of ORF1. Thus, we constructed the plasmid pGR3. This was designed so that the initiation codon is located at 19 base pairs downstream of the EcoRI site of pKK223-3 with an artificial ribosomebinding region (AGGA and CC next to ATG) (Fig. 2). We also carried out the site-directed mutagenesis study of (3-73 and C-184 of the P.pentosaceus enzyme and found that conversion of both cysteinyl residues to alanyl residues led to a loss of the enzyme activity.

--

AGCCA~A~ACCAGnCCATGAGACTCGGTC1TAAGTCCTGT'ITCTGGTACTCTG-

amp'

6.40 Kb

I

Hindlll

14 I- f

FIG.2. Construction of the plasmidspGRP and pGR3. The length of DNA fragments shown is arbitrary. 97,400-

'

E'

45,000 "+

31,500

-

21,500

-

14.400

-

9 A

B

C

D

FIG.3. SDS-PAGE of the cell extract and cell pellet extract of E. coli JM109/pGR3 and JM109/pKK223-3. Each cell extract (50 pg of protein) andcell pellet extract (30 pg of protein) were run on an SDS-PAGE (12.5% acrylamide) with the following molec= 97,400), bovine ular weight marker proteins: phosphorylase b (M, serum albumin (66,200), ovalbumin (45,000), bovine carbonic anhydrase (31,500), soybean trypsininhibitor (21,500), and lysozyme (14,400). Lane A, cell extract of E. coli JM109/pGR3; lane B, cell pellet extract of E. coli JM109/pGR3; lane C, cell pellet extract of E. coli JM109/pKK223-3; lane D,cell extract of E. coli JM109/pKK2233.

SDS-PAGE of the cell extract of E. coli JM109/pGR3 showed the occurrence of protein with a molecular weight of about 31,000 (Fig. 3, lane A ) . When the glutamate racemase of P.pentosaceus was overproduced under the control of tac promoter in the E. coli clone cells, it was found in inclusion bodies (16). We found the occurrence of protein with molecular weight of about 31,000 in the cell pellet extract of E. coli JM109/pGR3 (Fig. 3, lane B ) . The 31,000 protein band was found neither in the cell pellet extract nor in thecell extract of E. coli JM109/pKK223-3 (Fig. 3, lanes C and D). The glutamate racemase activity of extracts of E. coli JM109/pGR3 and JM109/pKK223-3 cells was determined

E. coli Glutamate Racemase with L-glutamate oxidase. Only the cell extract of JM109/ pGR3 showed the activity (specific activity, 0.53 units/mg of protein). The activity was detected in neither the cell pellet extract of JM109/pGR3 nor the extracts of E. coli JM109/ pKK223-3. The glutamate racemase activity was confirmed by high performance liquid chromatography after thereaction product was derivatized to form a diastereomer with Marfey's reagent. When D-glutamate was incubated with the cell extract of E. coli JM109/pGR3, L-glutamate was produced (Fig. 4). L-Glutamate was not formed in the reaction with the cell extract of E. coli JM1091pKK223-3. These results demonstrated that the murI gene encodes glutamate racemase, and its product is glutamate racemase. Accordingly, it is more relevant to designate the gene (dga) as glr than as m u d or dga, and thenew term, glr, is used hereafter. Molecular Weight of Glutamate Racemase Encoded by the glr Gene-Glutamate racemase encoded by the glr gene was purified about 4-fold as described above. A molecular weight of the purified enzyme was determined to be about 64,000 by means of gel filtration with a TSK-G~OOOSW column. SDSPAGE showed that each active fraction elutedfrom the TSKG3000sw column contained a proteinwith a molecular weight of 31,000 (data not shown). Thus, glutamate racemase of E. coli consists of two identical subunits encoded by the glr gene. Glutamate racemases of P . pentosaceus and L. fermenti are monomer enzymes (16), but their primary structures are homologous with that of the E. coli enzyme. This is the first example of the same kind of amino acid racemase having the distinct subunit structures. It is possible that only the E. coli enzyme has a composite active site formed at theinterface of two identical subunits in the same manner as that proposed for proline racemase (25). We are currently investigating the reaction mechanism of both enzymes comparatively. In this work, we have shown the glutamate racemase activity of the glr gene-encoding protein in E. coli JM109 cells transformed with the plasmid pGR3, which has a designed ribosome binding region. The structure of 5"upstream region of the open reading frame of glr probably does not fit the gene expression and is needed to be modified. The open reading frame of glr overlaps by the 66-base pair end of the preceding btuB gene (15, 26) and lacks adefinite ribosome binding

B

A

C

D

r

DGlu

D-Glu

D-GI" L-GIU

24245

region. Glutamate racemase has not been demonstrated in E. coli and various other bacteria (14) except lactic acid bacteria (7-11). The catalytic efficiency of glutamate racemase of P . pentosaceus is very low,about only 0.2% of that of the alanine racemase of Bacillus stearothermophilus (16, 27) and that of glutamate racemase of E. coli is probably low by analogy of the P . pentosaceus enzyme. This low catalytic efficiency and also probably the low expression efficiency of the glr gene may result in the failure of enzyme demonstration in E. coli (13). Alternatively, glutamate racemase may usually be produced only scarcely except that anappreciable amount of the enzyme is produced only on cell division. Incorporation of D-Glutamate, L-Glutamate, L-Proline, and a-Ketoglutarate into the Peptidoglycan of E. coli K-12 CellsWhen E. coli WM335 cells, a D-glutamate auxotroph having mutation in the glr gene, were cultured in the absence of Dglutamate, UDP-N-acetylmuramyl-L-alanine, a substrate of D-glutamate-adding enzyme, was accumulated (13). Thissuggests that the D-glutamate result from racemization of Lglutamate by glutamate racemase is incorporated into a peptidoglycan. We investigated the precursor of D-glutamyl residue of peptidoglycan by means of 14C-labeledamino acids and a-ketoglutarate. When E. coli K-12 cells were cultured in the M9 minimal medium with each 740 kBq (4.4 x lo7 dpm) of ~-[U-'~C]glutamate, ~-[U-'~C]glutamate, ~-[U-'~C]proline, or [U-'4C]a-ketoglutarate, 14C-labeledglutamate with the specific radioactivity of 8.4 x lo6 (dpm/pmol), 7.4 x lo4, 3.9 x lo4, 8.8 X lo4, respectively, were found in the peptidoglycan of cells. Higher incorporation of the radioactivity from Dglutamate confirms that D-glutamate is a direct precursor of the glutamyl residue of peptidoglycan. Radioactivity incorporated from L-glutamate into the glutamyl residue of peptidoglycan wasabout two times higher than thatfrom L-proline and nearly the same as that from a-ketoglutarate. Racemization of L-glutamate is thus arate-limitingstep of the formation of D-glUtamate in peptidoglycan. The results obtainedin this studyindicate that D-glutamate is produced from its L-enantiomer by glutamate racemase encoded by the glr gene in E . coli. D-Glutamate is indispensable for almost all of the bacteria as a constituent of peptidoglycan, but its physiological role is not known in mammals and other organisms than usual bacteria. Glutamate racemase thus can possibly be a target of antibiotic action. We are now investigating the detailed properties and structure of the E. coli enzyme. Acknowledgments-We thank Dr. Y. Kohara and Professor A. Ishihama of the National Institute of Genetics, Japan, for providing us with XEllCll andDr. H. Kusakabe of Yamasa Shoyu for supplying us with L-glutamate oxidase.

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REFERENCES 1. Mengin-Lecreulx, D. C., Parquet, L. R., Desviat, J., Pla, B., Flouret, J. A.,

u 50

60 70 (min)

- 50

60 70 (dn)

50

bo

(min)

70

h 50

60 70

(dn)

FIG.4. Conversion of D-glUtamatetoL-glutamate by the cell extract of E. coli JM109/pGR3. The reaction mixture contained 9.2 mg (as protein) of cell extract of E. coli JM109/pGR3 or 12 mg (as protein) of cell extract of E. coli JM109/pKK223-3. A , authentic D- and L-glutamate; B , reaction with cell extract of JM109/ pGR3, zero time; C,reaction with cell extract of E. coli JM109/pGR3, 2 h; D,reaction with cell extract of E. coli JM109/pKK223-3, 2 h.

Ayala, J., and van Heijenoort, J. (1989) J. Bacteriol. 176, 6126-6134 2. Thorne, C. B., Gornez, C. G., and Housewright, R. D. (1955) J. Bacteriol. 69,357-362 3. Thorne, C. B., and Molnar, D. M. (1955) J.Bacterid. 7 0 , 420-426 4. Meadow, P., and Work, E. (1958) Biochim. Biophys. Acta 524.60-67 5. Kuramitsu, H. K., and Snoke,J. E. (1962) Biochim. Biophys.Acta 62,114-

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