Publications

• Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus.

  Kim S.S., Choi I.G., Kim S.H., Yu Y.G.

  A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis ... >> More

  A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts L- or D-glutamate to D- or L-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The Km and k(cat) values of the overexpressed A. pyrophilus glutamate racemase for the racemization of L-glutamate to the D-form and the conversion of D-glutamate to the L-form were measured as 1.8 +/- 0.4mM and 0.79 +/- 0.06s(-1) or 0.50 +/- 0.07mM and 0.25 +/-0.01s(-1), respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85 degrees C for 90 min. << Less

  Extremophiles 3:175-183(1999) [PubMed] [EuropePMC]

• Catalytic acid/base residues of glutamate racemase.

  Glavas S., Tanner M.E.

  Glutamate racemase is a cofactor-independent enzyme that employs two active-site cysteine residues as acid/base catalysts during the interconversion of glutamate enantiomers. In a given reaction direction, a thiolate from one of the cysteines abstracts the alpha-proton, and the other cysteine thio ... >> More

  Glutamate racemase is a cofactor-independent enzyme that employs two active-site cysteine residues as acid/base catalysts during the interconversion of glutamate enantiomers. In a given reaction direction, a thiolate from one of the cysteines abstracts the alpha-proton, and the other cysteine thiol delivers a proton to the opposite face of the resulting carbanionic intermediate. This paper reports that the C73S and C184S mutants are still capable of racemizing glutamate with specificity constants about 10(3)-fold lower than those of the wild-type enzyme. A "one-base requiring" reaction, the elimination of water from N-hydroxyglutamate, has been used to deduce which thiol acts as the base for a given enantiomer. With D-N-hydroxyglutamate the C73S mutant is a much poorer catalyst than wild-type enzyme, whereas the C184S mutant is a somewhat better catalyst. This trend was reversed with L-N-hydroxyglutamate, suggesting that Cys73 is responsible for the deprotonation of D-glutamate and Cys184 is responsible for the deprotonation of L-glutamate. In addition, with C73S the Vmax/KM isotope effect on D-glutamate racemization was greater than that seen with wild-type enzyme, whereas the isotope effect with L-glutamate had decreased. The results were reversed with the C184S mutant. This is interpreted as being due to an asymmetry in the free energy profiles that is induced upon mutation, with the deprotonation step involving a serine becoming the more cleanly rate-determining of the two. These results support the above assignment and the notion that a carbanionic intermediate is formed during catalysis. << Less

  Biochemistry 38:4106-4113(1999) [PubMed] [EuropePMC]

• Structure and mechanism of glutamate racemase from Aquifex pyrophilus.

  Hwang K.Y., Cho C.-S., Kim S.S., Sung H.-C., Yu Y.G., Cho Y.

  Glutamate racemase (MurI) is responsible for the synthesis of D-glutamate, an essential building block of the peptidoglycan layer in bacterial cell walls. The crystal structure of glutamate racemase from Aquifex pyrophilus, determined at 2.3 A resolution, reveals that the enzyme forms a dimer and ... >> More

  Glutamate racemase (MurI) is responsible for the synthesis of D-glutamate, an essential building block of the peptidoglycan layer in bacterial cell walls. The crystal structure of glutamate racemase from Aquifex pyrophilus, determined at 2.3 A resolution, reveals that the enzyme forms a dimer and each monomer consists of two alpha/beta fold domains, a unique structure that has not been observed in other racemases or members of an enolase superfamily. A substrate analog, D-glutamine, binds to the deep pocket formed by conserved residues from two monomers. The structural and mutational analyses allow us to propose a mechanism of metal cofactor-independent glutamate racemase in which two cysteine residues are involved in catalysis. << Less

  Nat. Struct. Biol. 6:422-426(1999) [PubMed] [EuropePMC]

• Exploitation of structural and regulatory diversity in glutamate racemases.

  Lundqvist T., Fisher S.L., Kern G., Folmer R.H., Xue Y., Newton D.T., Keating T.A., Alm R.A., de Jonge B.L.

  Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we de ... >> More

  Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents. << Less

  Nature 447:817-822(2007) [PubMed] [EuropePMC]

• Isotope effects and the identification of catalytic residues in the reaction catalyzed by glutamate racemase.

  Tanner M.E., Gallo K.A., Knowles J.R.

  Primary kinetic isotope effects on Vmax were observed in both reaction directions upon racemizing samples of [2-2H]glutamate with the cofactor-independent glutamate racemase from Lactobacillus. This supports a deprotonation/protonation mechanism for racemization in which the breaking of the carbon ... >> More

  Primary kinetic isotope effects on Vmax were observed in both reaction directions upon racemizing samples of [2-2H]glutamate with the cofactor-independent glutamate racemase from Lactobacillus. This supports a deprotonation/protonation mechanism for racemization in which the breaking of the carbon-hydrogen bond at C-2 is partially rate-determining. Substantial "overshoots" were observed when the time course of racemization of either enantiomer of glutamate was monitored using circular dichroism spectroscopy. This is consistent with a "two-base" mechanism accompanied by a kinetic isotope effect. "Competitive deuterium washout" experiments were used to measure kinetic isotope effects on Vmax/Km of 2.5 for (S)-glutamate and 3.4 for (R)-glutamate. The ratio of the notably different isotope effects was confirmed by "double competitive deuterium washout" experiments. Site-directed mutagenesis was used to generate the mutant C73A and C184A enzymes. In each case the mutant enzymes were inactive as racemases. The two mutant enzymes are, however, capable of catalyzing the elimination of HCl from opposite enantiomers of threo-3-chloroglutamic acid, a process that presumably requires only one enzymic base. This finding indicates that the active sites of the mutant enzymes are intact and that the two cysteines flank the bound substrate molecule. It appears that cysteine-73 is responsible for the abstraction of the C-2 hydrogen from (R)-glutamate and cysteine-184 abstracts the proton from (S)-glutamate in the racemization reaction of the wild-type enzyme. << Less

  Biochemistry 32:3998-4006(1993) [PubMed] [EuropePMC]

• Purification, cloning, and cofactor independence of glutamate racemase from Lactobacillus.

  Gallo K.A., Knowles J.R.

  Glutamate racemase has been purified more than 12,000-fold from Lactobacillus fermenti. The racemase gene has been cloned using standard hybridization techniques combined with a novel selection for in vivo glutamate racemase activity, and the racemase has been expressed in Escherichia coli as 20-2 ... >> More

  Glutamate racemase has been purified more than 12,000-fold from Lactobacillus fermenti. The racemase gene has been cloned using standard hybridization techniques combined with a novel selection for in vivo glutamate racemase activity, and the racemase has been expressed in Escherichia coli as 20-25% of the total soluble cell protein. The cloned gene product is indistinguishable from that purified from Lactobacillus and is a monomer of M(r) 28,300. Both a coupled enzymatic assay and a circular dichroism assay show that the enzyme follows Michaelis-Menten kinetics, with a Km of 0.3 mM and a kcat of 70 s-1 in each reaction direction. Investigations into the cofactor dependence of glutamate racemase indicate that the enzyme employs neither pyridoxal phosphate nor a pyruvoyl group in the labilization of the proton at the stereogenic center of glutamate. Furthermore, the racemase activity is unaffected by the presence of the metal-chelating reagent EDTA. The gene sequence of the racemase is 24% identical to that of aspartate racemase from Streptococcus thermophilus and 30% identical to that of an unidentified open reading frame in the rrnB ribosomal RNA operon of E. coli. Because the two cysteine residues in glutamate racemase and their surrounding regions are well-conserved in both of these sequences, and since glutamate racemase is stabilized by the presence of reduced thiols, these residues are possible candidates for the enzymic bases that deprotonate glutamate at C-2. << Less

  Biochemistry 32:3981-3990(1993) [PubMed] [EuropePMC]

• Glutamic acid racemase from Lactobacillus arabinosus.

  GLASER L.

  J Biol Chem 235:2095-2098(1960) [PubMed] [EuropePMC]

• Effects of the S2-serotonergic receptor antagonist, ketanserin, on cerulein-induced pancreatitis in the rat.

  Oguchi H., Terashima M., Koiwai T., Kawa S., Furuta S., Kobayashi M., Homma T.

  We investigated the effects of ketanserin, a S 2 (5-hydroxytryptamine 2; 5-HT 2)-serotonergic receptor antagonist, on cerulein-induced pancreatitis in the rat. Large pharmacological doses of cerulein induced acute pancreatitis in the rat. Ketanserin reduced the cerulein-induced increase in serum a ... >> More

  We investigated the effects of ketanserin, a S 2 (5-hydroxytryptamine 2; 5-HT 2)-serotonergic receptor antagonist, on cerulein-induced pancreatitis in the rat. Large pharmacological doses of cerulein induced acute pancreatitis in the rat. Ketanserin reduced the cerulein-induced increase in serum amylase concentration in a dose-dependent manner. Treatment with 10 mg/kg of ketanserin per os markedly improved cerulein-induced pancreatitis and was associated with a significant reduction of the increase in serum amylase concentration. In addition, a very specific serotonin S 2 antagonist, ritanserin which has no antihypertensive effect, also reduced the cerulein-induced increase in the serum amylase concentration. These results suggest that S 2 (5-HT 2) may play a role in pathophysiology of cerulein-induced pancreatitis in the rat. << Less

  Life Sci 50:733-737(1992) [PubMed] [EuropePMC]