Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10144-10147, October 1994 Biochemistry Bacterial glutamate racemase has high sequence similarity with myoglobins and forms an equimolar inactive complex with hemin SOO-YOUNG CHOI*, NOBUYOSHI ESAKI, MAKOTO ASHIUCHI, TOHRU YOSHIMURA, AND KENJI SODAt Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan Communicated by Alton Meister, June 15, 1994 (receivedfor review January 3, 1994) ABSTRACT Glutamate racemase (EC 5.1.1.3), an enzyme with an Applied Biosystems model 470A gas-liquid-phase of microbial origin, shows siiicant sequence homology with protein sequencer. mammalian myoglobins, in particular in the regions corre- Stoichiometry of Hemin Binding with Glutamate Racemase. sponding to the E and F helices, which constitute the heme Various amounts of glutamate racemase (58, 116, 174, 232, binding pocket of myoglobins. Glutamate racemase binds and 290 nmol) were incubated in a solution (1.0 ml) containing tightly an equimolar amount of hemin, leading to loss of 3.2 mM hemin and 50 mM Tris+HCl (pH 7.5) at 25°C for 1 hr. racemase activity. Although this enzyme shows homology with This reaction solution was chromatographed on an Excellu- aspartate racemase, the latter does not bind hemin. The lose GF-5 column (Pierce) equilibrated with 50 mM Tris HCl, giutamate racemase gene of Pediococcus pentosaceus has a pH 7.5. Protein was eluted by an isocratic elution with the 795-nt open reading frame and encodes 265-amino acid resi- same solvent. Protein concentrations were determined with dues, which form a monomeric protein (Mr 29,000). Neither a Bio-Rad protein assay kit. Enzyme concentration was also racemase has cofactors, but they contain essential cysteine determined with 5,5'-dithiobis-2-nitrobenzoic acid: the en- residues [Yohda, M., Okada, H. & Kumagai, H. (1991) zyme contains three cysteinyl residues per mol. Enzyme- Biochim. Biophys. Acta 1089, 234-240]. bound hemin was determined by measurement of atomic absorption of iron with a Shimadzu model AA-670G atomic D-Glutamate is an essential component of peptidoglycans of absorption spectrophotometer equipped with a graphite fur- bacterial cell walls and is produced from L-glutamate by nace atomizer. An average of seven independent determina- glutamate racemase (EC 5.1.1.3) or from a-ketoglutarate by tions was taken. D-amino acid aminotransferase (EC 2.6.1.21) (1). Most amino acid racemases, such as alanine racemase (EC 5.1.1.1), require pyridoxal 5'-phosphate as a coenzyme (2), and the RESULTS racemase reaction is facilitated by formation of internal and DNA Sequencing and Primary Structure ofGlutamate Race- external Schiff base intermediates. In contrast, a few other mase. The DNA sequence of the 1.8-kb fragment derived amino acid racemases, such as glutamate racemase (3-6, 25, from pICR223 contained an open reading frame of795 bp, and 26) and aspartate racemase (EC 5.1.1.13) (7, 8), are indepen- the region encoded a protein of 265-amino acid residues with dent of any cofactor and contain no carbonyl moieties or apredicted Mr of29,143 (Fig. 1). This predicted weight agrees metals. Their reaction mechanisms have not been elucidated. well with the Mr 29,000 determined by SDS/gel electropho- We have cloned the glutamate racemase gene from Pedio- resis of the enzyme (9). coccus pentosaceus, expressed it in Escherichia coli, and Sequence Similarity with Other Amino Acid Racems. The purified the enzyme to homogeneity (3, 9). The purified deduced amino acid sequence of the P. pentosaceus gluta- enzyme contains no cofactors but does have essential cys- mate racemase was compared with those of pyridoxyl 5'- teine residues. phosphate-independent amino acid racemases and epime- We report here the primary structure of glutamate race- rases-namely, proline racemase (EC 5.1.1.4) (10, 11), 4-hy- maset and show that it has sequence homology with aspartate droxyproline epimerase (EC 5.1.1.8) (12), diaminopimelate racemase, in particular in the vicinity of the two cysteine epimerase (EC 5.1.1.7) (13, 14), and aspartate racemase (7, residues. Very interestingly, the sequence ofglutamate race- 15), as well as pyridoxyl 5'-phosphate-dependent alanine mase also shows high homology with bovine myoglobin. The racemases. Only aspartate racemase showed considerable inhibition of glutamate racemase by formation of an equimo- sequence homology with glutamate racemase. Linear align- lar complex with hemin is also reported. ment of their sequences by introducing gaps to maximize identity revealed an overall homology of 14% (Fig. 2). EXPERIMENTAL PROCEDURES However, sequence homology in the internal region (69-192 DNA Sequencing. A recombinant plasmid, pICR223, car- of the glutamate racemase sequence) was much higher, 31 of rying an insert of 1.8 kb coding for the glutamate racemase 124 residues being common. If the mutationally allowed gene was obtained as described (9). The 1.8-kb insert was substitutions for similar residues were considered, the sim- blunt-ended and ligated into the Sma I site of pUC118 and ilarity score increased to 68% in this region. In particular, the -119. The insert was then unidirectionally deleted to various sequences around the two cysteine residues (Cys-74 and lengths with a double-stranded nested deletion kit (Pharma- Cys-184 of glutamate racemase) were highly homologous. cia). The single-stranded DNA was isolated and sequenced Both enzymes contain an essential cysteine residue as re- by the modified Sanger method with an Applied Biosystems ported previously, suggesting that either Cys-74 or Cys-184, model 370A DNA sequencer. To identify the open reading or both play an essential role in catalysis. frame, we determined the N-terminal amino acid sequence of glutamate racemase, which was purified as described (9), *Present address: Department of Biological Chemistry and Molec- ular Pharmacology, Harvard Medical School, 250 Longwood Av- enue, Boston, MA 02115. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L22448). 10144 Downloaded by guest on October 1, 2021 Biochemistry: Choi et al. Proc. Natl. Acad. Sci. USA 91 (1994) 10145 60 AT2.GAT .AATM.CUT.CCA.ATT GO00 TT22 AT9.GAT. TCA.SGC.OTC.0T2.0GOT.CTG.ACA.GTT .GTC.AAA M-t Asp Asn Arg Pro Ile Gly Phe MNt Asp 8er Gly Val Oly Gly Lou Thr Val Val Lys 120 ACA.OCT .CAA.AAA. CT0 . TTA. CCT .AAT . aAA. GAA.ATT.ATC . TS2 .ATT .caa.OAT . GAA.GCA.AaA.ATa Thr Ala Gin Lys Lou Lou Pro Ann Olu Olu Ile Ile Pho Ile Gly Asp Olu Ala Arg Met 180 CCG. TAT . 002. CCT .CGT . CCC . ACA. GCA. GAA. GTC . 0TT .GAA. TTT . TCA. C0A. CAG .ATa . OCO. TCA. TTT Pro Tyr Gly Pro Arg Pro Thr Ala Glu Val Val Glu Phe Ser Arg Gin met Ala 8-r Ph. 240 TTA.ATG . ACT .AAA.AAT .ATT .AAG. OCo . CTA. 0T2 .ATT GCA. TOT . AAT .ACT . GCG . ACC . AAGCG..CG Lou Met Thr Lys Asn Ile Lys Ala Lou Val I1e Ala Cys Asn Thr Ala Thr Asa Ala Ala 300 2TA. GCG.GTT. STA. CAA.OCT . GA. 2A£. CCC .ATC . CCA.TA.ATT .0G . GT0.ATT . TTA.CCT .GGC .GCA Lou Ala Val Lou gin Ala Glu Lou Pro Ile Pro Val Ile Gly Val Ile Lou Pro Gly Ala 360 TT .GCA.OCT .A2 .aGa .CAA.ACT . AAA. aAT. CAa.AAAA .ATC . GA.GT.aTT .OCT . ACA. CTA. GC .ACA Iie Ala Ala Asn Arg Gin Thr Lys Asn Gin Lys Ile Gly Val Ile Ala Thr Lou gly Thr 420 £22 .AAA. TCT . G.AG OCT. TAC . CCA.AAG . OCT. TTA. OCT .GAA.ATT .AAT .ACC .AAA. TTA.CgT. OCT . TAT Ile Lys 8er Glu Ala Tyr Pro Lys Ala Lou Ala Glu Ile Asa Thr Lys Lou Arg Ala Tyr 480 CCG . aTa. CCA. TGC . CAA. GAA. TTT2.TA. GAA.ATT . OCT . GAA.AAA. AAT . GAA. CTT . CAT .ACA.ACG . OCA Pro Val Ala Cys Gin Glu Phe Val Giu Ile Ala Glu Lys Asna lu Lou His Thr Thr Ala 540 OCT .CAA.AAA. GTT .ATO .AAT .GAA. AAA. CTA.OCT . 2a.TA002 . CAA. GAT .CAA.ATS .GAT.ACT .2T2 Ala Gin Lys Via Met Asa Glu Lys Lou Ala Gia Ph. Arg Gin Asp Gin Ile Asp Thr Lou 600 aTT2 .TTA.Gac . TOC .ACT .CAT. 2TT. CCG. CTT . TTA2.Aa. GAI. 002 .ATC . CAA. aCA.OCCC . TTGG.0.CCT I1e Lou 0ly Cys Thr His Phe Pro Lou Lou Glu Glu Gly Ile Gin Ala Ala Val Gly Pro 660 OAT . T . ACT. TA£. GA tGAT . CCA. 00A. GT02 GAG .ACC. 0TT2. CAc.CAA. TTA.AT2 (M. ATT .TTA. ACA Asp Val Thr Lou Val Asp Pro Gly Val Glu Thr Val His Gin Lou Ile Glu Ile Lou Thr 720 a£a. CAA.aCG .CTT . CAa.CAT .aCA.OAA. aT2. CCT .AAA.OCG .CAO . OAT .CAa. TAT . TAT. TCA.ACG .002 Lys Gin Ala Lou Gin His Ala Glu Gly Pro Lys Ala Gin Asp gin Tyr Tyr 8-r Thr Gly 780 A£T .AT .AG.AA2 222. GAl. GAA.ATA.GCG. C.ACA.TTC . A.AAT .CAA. GAT.CTA.AGI.GT .GA Asn IIe Lys Asn Ph. Glu Glu Ile Ala Arg Thr Ph. Lou Asn Gin Asp Lou Arg Val Glu 798 GAA.oTT*AAA.ATT.GAC.TAG Giu Val Lys I1i Asp Stop FIG. 1. DNA sequence of the glutamate racemase gene and the deduced amino acid sequence of the enzyme.