Caractérisation génétique et biochimique du système de racémisation du lactate chez Lactobacillus plantarum
(2013)
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- (en) Lactate is ubiquitous in nature and is involved in the energy metabolism of many prokaryotic species, as a product of sugar fermentation or as a carbon and electron source to sustain growth. The lactate racemase (Lar) converts L- or D- lactate in a racemic mixture of DL-lactate, giving micro-organisms the ability to metabolize both lactate isomers. The biochemistry of the Lar enzyme has remained largely unexplored due to a lack of molecular characterization. The locus responsible for this activity in Lactobacillus plantarum was previously identified by our laboratory. This locus, named lar, is composed of two operons (larR(MN)OQ and larABCDE). In this thesis, we report that four proteins, LarA-B-C-E, are absolutely required for lactate racemization. LarA was identified as the racemase and nickel was found as an essential cofactor of the enzyme. LarA is therefore the first racemase entering the group of nickel-dependent enzymes (9 members now). The purified enzyme was biochemically characterized and inhibitors were identified. The enzymes 3D structure revealed a novel α/β fold. Based on these results and previous biochemical data, we proposed a catalytic mechanism that involves Ni-mediated hydride abstraction. We also demonstrated that the three LarB-C-E proteins act as an original nickel-carrying maturation machinery necessary for LarA activation. In vitro activation results indicated a mechanism in which LarE activates LarA and is activated by LarB-C. We characterized LarD, which transports lactic acid, thereby broadening the range of substrates transported by bacterial aquaglyceroporins. We also investigated the transcriptional regulator LarR, responsible for the regulation of the lar locus depending on the L-/D-lactate ratio. Finally, a bioinformatic analysis revealed the presence of lactate racemase genes in nearly a tenth of all prokaryotic genomes examined, suggesting the involvement of lactate racemization in the metabolism of a wide variety of species.
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Desguin, B. (2013). Caractérisation génétique et biochimique du système de racémisation du lactate chez Lactobacillus plantarum. https://hdl.handle.net/2078.5/203589