The enzyme has been characterized from the bacterium Lactobacillus plantarum and appears to be restricted to lactic acid bacteria. It contains a unique nickel-containing cofactor, pyridinium-3-thioamide-5-thiocarboxylate mononucleotide Ni pincer complex.
the enzyme does not follow a proton-coupled electron transfer mechanism, but a proton-coupled hydride transfer (PCHT) mechanism. The nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization
the Ni-tethered pincer cofactor in enzyme LarA increases reaction barriers and destabilizes NADH-like pyruvate intermediates, due to the less electrophilic Ni cofactor and to the ring strain in the pyruvate intermediates, reaction mechanism, active and transition state structure, modeling
the enzyme does not follow a proton-coupled electron transfer mechanism, but a proton-coupled hydride transfer (PCHT) mechanism. The nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization
the Ni-tethered pincer cofactor in enzyme LarA increases reaction barriers and destabilizes NADH-like pyruvate intermediates, due to the less electrophilic Ni cofactor and to the ring strain in the pyruvate intermediates, reaction mechanism, active and transition state structure, modeling
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SYSTEMATIC NAME
IUBMB Comments
lactate racemase
The enzyme has been characterized from the bacterium Lactobacillus plantarum and appears to be restricted to lactic acid bacteria. It contains a unique nickel-containing cofactor, pyridinium-3-thioamide-5-thiocarboxylate mononucleotide Ni pincer complex.
the enzyme contains an organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide. The nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization
the enzyme contains an organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide. The nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization
absolutely required. Lactobacillus plantarum possesses an organometallic nickel-containing prosthetic group. A nicotinic acid mononucleotide derivative is tethered to Lys184 and forms a tridentate pincer complex that coordinates nickel through one metal-carbon and two metal-sulfur bonds, with His200 as another ligand. Nickel-binding site structure and the role of three accessory proteins required for its activation, overview
dependent on, nickel is an essential cofactor, the enzyme contains 19-21% nickel, measured by PAR assays and ICP-AES. The LarA Ni center is coordinated by His residues, a four coordinate square planar nickel center or a five coordinate square pyramidal site
required, the Ni-tethered pincer cofactor in enzyme LarA increases reaction barriers and destabilizes NADH-like pyruvate intermediates, due to the less electrophilic Ni cofactor and to the ring strain in the pyruvate intermediates, the Ni ion decreases the electron affinity of cofactor
model of the assembly of the lactate racemase metallocenter, overview. Enzyme LarA receives Ni2+ from the pyridinium-3,5-bisthiocarboxylic acid mononucleotide synthase, LarE (EC 4.4.1.37)
model of the assembly of the lactate racemase metallocenter, overview. Enzyme LarA receives Ni2+ from the pyridinium-3,5-bisthiocarboxylic acid mononucleotide synthase, LarE (EC 4.4.1.37)
hypothetical model of PlarA regulation by LarR: in the presence of L-lactate, activated LarR binds to the Lar box motif and multimerizes on the half-Lar boxes. This will promote direct interaction of one LarR dimer with the RNA polymerase, resulting in transcriptional activation of the PlarA (productive binding). In the presence of D-lactate, D-lactatet can block LarR activation, for instance, by impairing L-lactate recognition, which will result in limited LarR binding and multimerization and absence of transcriptional activation (unproductive binding)
lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system. Effect of accessory Lar proteins and cofactors on the in vitro activation of LarANiDELTABCE (apo-LarA) by LarENiBC, overview. Necessity of the Lar accessory proteins (larBCE), for LarA activation. Ni-loaded LarE acts as a maturation protein responsible for the activation of apo-LarA, and indicate that LarB and LarC are involved in the activation of LarE prior to apo-LarA activation
lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system. Effect of accessory Lar proteins and cofactors on the in vitro activation of LarANiDELTABCE (apo-LarA) by LarENiBC, overview. Necessity of the Lar accessory proteins (larBCE), for LarA activation. Ni-loaded LarE acts as a maturation protein responsible for the activation of apo-LarA, and indicate that LarB and LarC are involved in the activation of LarE prior to apo-LarA activation
the lactate racemase is a nickel-dependent enzyme requiring activation by the accessory protein LarE, which itself requires activation by the accessory proteins LarB and LarC and nickel
the lactate racemase is a nickel-dependent enzyme requiring activation by the accessory protein LarE, which itself requires activation by the accessory proteins LarB and LarC and nickel
analysis of the lar gene cluster and its encoded Lar proteins, phylogenetic tree. 92% of the genomes bearing a larA homologue (102 out of 111 genomes) also contain the genes for the Lar accessory proteins (larBCE), further reinforcing the necessity of these proteins for LarA activation
analysis of the lar gene cluster and its encoded Lar proteins, phylogenetic tree. 92% of the genomes bearing a larA homologue (102 out of 111 genomes) also contain the genes for the Lar accessory proteins (larBCE), further reinforcing the necessity of these proteins for LarA activation
disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between D- and L-latate, completely abolishes D-lactate production. An engineered Lactobacillus plantarum strain lacking the enzyme is useful in the production of L-lactate from starchy materials
disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between D- and L-latate, completely abolishes D-lactate production. An engineered Lactobacillus plantarum strain lacking the enzyme is useful in the production of L-lactate from starchy materials
lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system. Four proteins and Ni are required for in vivo Lar activity. Necessity of the Lar accessory proteins (larBCE), for LarA activation. Ni-loaded LarE acts as a maturation protein responsible for the activation of apo-LarA, and indicate that LarB and LarC are involved in the activation of LarE prior to apo-LarA activation
the lactate racemase is a nickel-dependent enzyme requiring activation by the accessory protein LarE, which itself requires activation by the accessory proteins LarB and LarC and nickel. The interconversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by the L-/D-lactate ratio and maximally induced in the presence of L-lactate. The Lar activity depends on the expression of two divergently oriented operons: (i) the larABCDE operon encodes the nickel-dependent lactate racemase (LarA), its maturases (LarBCE), and a lactic acid channel (LarD), and (ii) the larR(MN)QO operon encodes a transcriptional regulator (LarR) and a four-component ABC-type nickel transporter [Lar(MN), in which the M and N components are fused, LarQ, and LarO]. LarR is a regulator of the Crp-Fnr family (PrfA group). L-Lactate has a positive effect on the binding and multimerization of LarR, while D-lactate antagonizes the positive effect of L-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed. Hypothetical model of PlarA regulation by LarR: in the presence of L-lactate, activated LarR binds to the Lar box motif and multimerizes on the half-Lar boxes. This will promote direct interaction of one LarR dimer with the RNA polymerase, resulting in transcriptional activation of the PlarA (productive binding). In the presence of D-lactate, D-lactatet can block LarR activation, for instance, by impairing L-lactate recognition, which will result in limited LarR binding and multimerization and absence of transcriptional activation (unproductive binding). Role of LarR in vivo and in vitro
lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system. Four proteins and Ni are required for in vivo Lar activity. Necessity of the Lar accessory proteins (larBCE), for LarA activation. Ni-loaded LarE acts as a maturation protein responsible for the activation of apo-LarA, and indicate that LarB and LarC are involved in the activation of LarE prior to apo-LarA activation
the lactate racemase is a nickel-dependent enzyme requiring activation by the accessory protein LarE, which itself requires activation by the accessory proteins LarB and LarC and nickel. The interconversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by the L-/D-lactate ratio and maximally induced in the presence of L-lactate. The Lar activity depends on the expression of two divergently oriented operons: (i) the larABCDE operon encodes the nickel-dependent lactate racemase (LarA), its maturases (LarBCE), and a lactic acid channel (LarD), and (ii) the larR(MN)QO operon encodes a transcriptional regulator (LarR) and a four-component ABC-type nickel transporter [Lar(MN), in which the M and N components are fused, LarQ, and LarO]. LarR is a regulator of the Crp-Fnr family (PrfA group). L-Lactate has a positive effect on the binding and multimerization of LarR, while D-lactate antagonizes the positive effect of L-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed. Hypothetical model of PlarA regulation by LarR: in the presence of L-lactate, activated LarR binds to the Lar box motif and multimerizes on the half-Lar boxes. This will promote direct interaction of one LarR dimer with the RNA polymerase, resulting in transcriptional activation of the PlarA (productive binding). In the presence of D-lactate, D-lactatet can block LarR activation, for instance, by impairing L-lactate recognition, which will result in limited LarR binding and multimerization and absence of transcriptional activation (unproductive binding). Role of LarR in vivo and in vitro
electron paramagnetic resonance spectroscopy of LarA in the absence or presence of substrate revealing a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand
electron paramagnetic resonance spectroscopy of LarA in the absence or presence of substrate revealing a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand
electron paramagnetic resonance spectroscopy of LarA in the absence or presence of substrate revealing a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified enzyme, free or Hg-labeled, hanging drop vapour diffusion method, mixing of 0.002 ml of 16 mg/ml protein solution with 0.002 ml of reservoir solution containing 22% PEG monomethyl ether 5000, 0.2 M ammonium sulfate, 0.1 M MES pH 6.5, 0.2 M sodium malonate pH 7.0 and 0.02% w/v sodium azide, at 18°C, a few days, X-ray diffraction structure determination and analysis at 1.8-2.0 A resolution
construction of an engineered Lactobacillus plantarum strain NCIMB 8826, which enables the production of optically pure L-lactate from raw starch for costeffective lactate production. Disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between D- and L-lactate, completely abolishes D-lactate production. The DELTAldhD DELTAlarA-E mutant produces 87.0 g/l of L-lactate with an optical purity of 99.4%. A plasmid is introduced into the DELTAldhD DELTAlarA-E mutant for the secretion of alpha-amylase from Streptococcus bovis strain 148. The resulting strain produces 50.3 g/l of L-lactate from raw corn starch with a yield of 0.91 g per g of consumed sugar and an optical purity of 98.6%
construction of an engineered Lactobacillus plantarum strain NCIMB 8826, which enables the production of optically pure L-lactate from raw starch for costeffective lactate production. Disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between D- and L-lactate, completely abolishes D-lactate production. The DELTAldhD DELTAlarA-E mutant produces 87.0 g/l of L-lactate with an optical purity of 99.4%. A plasmid is introduced into the DELTAldhD DELTAlarA-E mutant for the secretion of alpha-amylase from Streptococcus bovis strain 148. The resulting strain produces 50.3 g/l of L-lactate from raw corn starch with a yield of 0.91 g per g of consumed sugar and an optical purity of 98.6%
construction of an engineered Lactobacillus plantarum strain NCIMB 8826, which enables the production of optically pure L-lactate from raw starch for costeffective lactate production. Disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between D- and L-lactate, completely abolishes D-lactate production. The DELTAldhD DELTAlarA-E mutant produces 87.0 g/l of L-lactate with an optical purity of 99.4%. A plasmid is introduced into the DELTAldhD DELTAlarA-E mutant for the secretion of alpha-amylase from Streptococcus bovis strain 148. The resulting strain produces 50.3 g/l of L-lactate from raw corn starch with a yield of 0.91 g per g of consumed sugar and an optical purity of 98.6%
recombinant StrepII-tagged LarA from Lactobacillus lactis by affinity chromatography, gel filtration, and ultrafiltration, to homogeneity. Purification of LarA and LarE can readily be achieved from the strain expressing the entire operon, whereas tagged LarB and LarC can only be purified when the corresponding genes are individually subcloned
gene larA, encoded in the lar operon, clustering of lar genes and genotyping, overview. Recombinant expression of N- or C-terminally StrepII-tagged LarA in Lactobacillus lactis
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
lactate racemase (Lar) is transcriptionally controlled by the L-/D-lactate ratio and maximally induced in the presence of L-lactate. LarR is a positive regulator that is absolutely required for the expression of Lar activity. LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in the larRlarA intergenic region
lactate racemase (Lar) is transcriptionally controlled by the L-/D-lactate ratio and maximally induced in the presence of L-lactate. LarR is a positive regulator that is absolutely required for the expression of Lar activity. LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in the larRlarA intergenic region
lactate racemase (Lar) is transcriptionally controlled by the L-/D-lactate ratio and maximally induced in the presence of L-lactate. LarR is a positive regulator that is absolutely required for the expression of Lar activity. LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in the larRlarA intergenic region
Untersuchungen zur Entstehung von DL-Milchsure bei Lactobacillen und Charakterisierung einer Milchsureracemase bei einigen Arten der Untergattung Streptobacterium
Diversity of lactate metabolism in halophilic archaea
Can. J. Microbiol.
41
302-307
1995
Haloarcula marismortui, Haloarcula vallismortis, Haloferax volcanii, no activity in Halobacterium saccharovorum, no activity in Halobacterium salinarum, no activity in Haloferax mediterranei