Lactate racemase

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Lactate racemase
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EC no. 5.1.2.1
CAS no. 2602118
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The lactate racemase enzyme (Lar) (EC 5.1.2.1) interconverts the D- and L-enantiomers of lactic acid. It is classified under the isomerase, racemase, epimerase, and enzyme acting on hydroxyl acids and derivatives classes of enzymes. [1] It is found in certain halophilic archaea, such as Haloarcula marismortui, and in a few species of bacteria, such as several Lactobacillus species (which produce D- and L-lactate) including Lactobacillus sakei, Lactobacillus curvatus, and Lactobacillus plantarum, as well as in non-lactic acid bacteria such as Clostridium beijerinckii. [2] The gene encoding lactate racemase in L. plantarum was identified as larA and shown to be associated with a widespread maturation system involving larB, larC1, larC2, and larE. [3] The optimal pH for its activity is 5.8-6.2 in L. sakei. [4]

Contents

Structure and properties

The molecular weight of lactate racemase differs in the various organisms in which it has been found, ranging from 25,000 to 82,400 g/mol. [5] The structure of the enzyme from L. plantarum was solved by Jian Hu and Robert P. Hausinger of Michigan State University and co-workers there and elsewhere. [6] The protein contains a previously unknown covalently-linked nickel-pincer nucleotide (NPN) cofactor (pyridinium 3-thioamide-5-thiocarboxylic acid mononucleotide), where the nickel atom is bound to C4 of the pyridinium ring and two sulfur atoms. This cofactor participates in a proton-coupled hydride-transfer mechanism. [7]

There have been a number of recent studies on NPN cofactor synthesis by the LarB, LarE, and LarC proteins. LarB is a carboxylase/hydrolase of nicotinamide adenine dinucleotide (NAD), providing pyridinium-3,5-dicarboxylic acid mononucleotide and adenosine monophosphate (AMP). [8] LarE is an ATP-dependent sulfur transferase that converts the two substrate carboxyl groups into thioacids by sacrificing the sulfur atoms of a cysteine residue in the protein. [9] Finally, LarC inserts nickel into the organic ligand by a CTP-dependent process to complete synthesis of the NPN cofactor. [10]

Enzyme activity

In many of the species containing lactate racemase, the physiological role of the enzyme is to convert substrate D-lactate into L-lactate. In other species, such as L. plantarum, the cellular role is to transform L-lactate into D-lactate for incorporation into the cell wall. [2]

The in vitro reaction catalyzed by the enzyme reaches equilibrium at the point where approximately equimolar concentrations of the D- and L-isomers exist. [4]

L. plantarum initially produces L-lactate, which induces the activity of lactate racemase. By contrast, D-lactate represses lactate racemase activity in this species. Therefore, Lar activity appears to be regulated by the ratio of L-lactate/D-lactate. L. plantarum LarA represents a new type of nickel-dependent enzyme, due to its novel nickel-pincer ligand ligand cofactor. [6]

Importance

Two pathways appear to exist in L. plantarum for transforming pyruvate into D-lactate. One of them involves the NAD-dependent lactate dehydrogenase that directly produces D-lactate (LdhD), and the other is through the sequential activities of an L-specific lactate dehydrogenase followed by lactate racemase. If the LdhD enzyme is inactivated or inhibited, lactate racemase provides the bacterium with a rescue pathway for the production of D-lactate. [2] This pathway is significant because the production of D-lactate in L. plantarum is linked to the biosynthesis of the cell wall. Mutants lacking LdhD activity that also had the lar operon deleted only produced L-lactate, and peptidoglycan biosynthesis was not able to occur.

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References

  1. ↑ "DBGET Result: ENZYME 5.1.2.1" . Retrieved 2007-06-03.
  2. 1 2 3 Goffin P; Deghorain M; Mainardi J-L; et al. (2005). "Lactate racemization as a rescue pathway for supplying D-lactate to the cell wall biosynthesis machinery in Lactobacillus plantarum". J. Bacteriol. 187 (19): 6750–61. doi:10.1128/JB.187.19.6750-6761.2005. PMC   1251571 . PMID   16166538.
  3. ↑ Desguin B, Goffin P, Viaene E, Kleerebezem M, Martin-Diaconescu V, Maroney MJ, Declercq JP, Soumillion P, Hols P (2014). "Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system". Nat. Commun. 5: 3615. doi: 10.1038/ncomms4615 . PMC   4066177 . PMID   24710389.
  4. 1 2 Hiyama T, Fukui S, Kitahara K (1968). "Purification and Properties of Lactate Racemase from Lactobacillus sake". J. Biochem. 64 (1): 99–107. doi:10.1093/oxfordjournals.jbchem.a128870. PMID   5707819.
  5. ↑ "BRENDA: Entry of Lactate racemase(EC-Number 5.1.2.1 )". Archived from the original on 2016-03-03. Retrieved 2007-06-03.
  6. 1 2 Desguin B, Zhang T, Soumillion P, Hols P, Hu J, Hausinger RP (2015). "A tethered niacin-derived pincer complex with a nickel-carbon bond in lactate racemase". Science. 349 (6243): 66–69. doi:10.1126/science.aab2272. PMID   26138974. S2CID   206637903.
  7. ↑ Rankin JA, Mauban RC, Fellner M, Desguin B, McCracken J, Hu J, Varganov SA, Hausinger RP (2018). "Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism". Biochemistry. 57 (23): 3244–3251. doi:10.1021/acs.biochem.8b00100. OSTI   1502215. PMID   29489337.
  8. ↑ Desguin B, Soumillion P, Hols P, Hausinger RP (2016). "Nickel-pincer cofactor biosynthesis involves LarB-catalyzed pyridinium carboxylation and LarE-dependent sacrificial sulfur insertion". Proc. Natl. Acad. Sci. USA. 113 (20): 5598–5603. doi: 10.1073/pnas.1600486113 . PMC   4878509 . PMID   27114550.
  9. ↑ Fellner M, Rankin JA, Desguin B, Hu J, Hausinger RP (2018). "Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum". Biochemistry. 57 (38): 5513–5523. doi:10.1021/acs.biochem.8b00601. OSTI   1476089. PMID   30157639. S2CID   52117187.
  10. ↑ Desguin B, Fellner M, Riant O, Hu J, Hausinger RP, Hols P, Soumillion P (2018). "Biosynthesis of the nickel-pincer nucleotide cofactor of lactate racemase requires a CTP-dependent cyclometallase". J. Biol. Chem. 293 (32): 12303–12317. doi: 10.1074/jbc.RA118.003741 . PMC   6093250 . PMID   29887527.
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