Characterization of acetyl-CoA and propionyl-CoA carboxylases encoded by Leptospira interrogans serovar Lai： an initial biochemical study for leptospiral gluconeogenesis via anaplerotic CO2 assimilation

Leptospira interrogans is the causative agent of leptospirosis. The in vitro growth of L. interrogans requires CO2 and a partial 3-hydroxypropionate pathway involving two aeyl-CoA carboxylases was suggested by genomic analysis to assimilate CO2. Either set of the candidate genes heterologously co-expressed in Escherichia coli was able to demonstrate both aeetyl-CoA carboxylase （ACC） and propionyi-CoA carboxylase （PCC） activities. The trisubunit holoenzyme （LA_2736-LA 2735 and LA 3803）, although failed to be purified, was designated ACC based on its substrate preference toward acetyl-CoA. The partially purified bi-subunit holoenzyme （LA_2432- LA_2433） has a considerably higher activity against pro- pionyl-CoA as the substrate than that of acetyl-CoA, and thus, designated PCC. Native polyacrylamide gel electro- phoresis indicated that this PCC has a molecular mass of around 669 kDa, suggesting an α4β4 quaternary structure and both structural homology modeling and site-directed mutagenesis analysis of its carboxyltransferase subunit （LA_2433） indicated that the A431 residue located at the bottom of the putative substrate binding pocket may play an important role in substrate specificity determination. Both transcriptomic and proteomic data indicated that enzymes involved in the suggested partial 3-hydroxypro- pionate pathway were expressed in vivo in addition to ACC/PCC and the homologous genes in genomes of other Leptospira species were re-annotated accordingly. However, as the in vitro detected specific activity of ACC in the crude cell extract was too low to account for the growth of the bacterium in Ellinghausen-McCuliough- Johnson-Harris minimal medium, further systematic analysis is required to unveil the mechanism of gluconeo- genesis via anaplerotic C02 assimilation in Leptospira species.