In vitro characterisation of lactate racemase homologues : A focus on three wide-spectrum α-hydroxy acid racemases and epimerases from Enterocloster asparagiformis

Details

Supervisors

Desguin, Benoît

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

Among the key enzymatic group of racemases and epimerases, there are racemases and epimerases acting on α-hydroxy acids (AHAs). Within this class, the lactate racemase (LarA) of Lactiplantibacillus plantarum was first characterised in detail. LarA’s activity was found to be dependent on a nickel-based pincer cofactor called the Nickel-Pincer Nucleotide (NPN). The genes encoding the LarA apoenzyme and the three biosynthetic enzymes for NPN biosynthesis, LarB, LarC and LarE, are found within the same operon in L. plantarum. Genes encoding homologues of larA (larAHs) are widely present in prokaryotes, with LarAHs forming a superfamily of NPN-dependent racemases and epimerases. Since these racemases and epimerases act on similar substrates as LarA, LarA’s mechanism can probably be generalised to all LarAHs. The characterisation of LarAHs had already begun before this thesis with the discovery of 7 LarAHs catalysing 5 new AHA racemisation and epimerisation reactions in 2020: two malate racemases, one phenyllactate racemase, one α-hydroxyglutarate racemase, two D-gluconate 2-epimerases, and one short-chain aliphatic α-hydroxy acid racemase. This thesis continues the in vitro characterisation of the LarAH superfamily. In the first part of this thesis, we biochemically characterised LarAH32, a previously identified α-hydroxyglutarate racemase from Geobacter metallireducens. The optimal pH and temperature as well as kinetic parameters (KM, kcat and kcat/KM) of this enzyme were determined with L-α-hydroxyglutarate. Then, we explored the substrate scopes of three enzymes from unknown groups of the LarAH superfamily: LarAH62, LarAH66 and LarAH67 from Sporomusa termitida. Though many substrates are still yet to be tested with these enzymes, we detected multiple activities for each of the three enzymes. Finally, we focused on three enzymes from Enterocloster asparagiformis, a bacterium found in the human gut microbiota. The studied enzymes, LarAH41, LarAH44 and LarAH49, belong to a group of wide-spectrum AHA racemases and epimerases. The optimal pH and temperature were determined for LarAH44. The kinetic parameters of LarAH41 and LarAH44 were determined with different substrates. We investigated the stability of LarAH41 at room temperature. Finally, considering the wide substrate spectra of the three studied enzymes, we examined their substrate preferences. Strikingly, the two preferred substrates for all three enzymes were found to be 2-hydroxy-4-phenylbutyrate and α-hydroxyisocaproate, which we postulate to be the native substrates of LarAH41, LarAH44, and LarAH49. Overall, this thesis contributes to the ever-growing understanding of the LarAH superfamily, which could give rise to biotechnological applications as well as progress in the understanding of microbial metabolism and its potential influence on the human health.