Characterization of recombinant homocitrate synthase from Candida albicans

LYS21 and LYS22 genes from Candida albicans encoding isoforms of homocitrate synthase (HCS), an enzyme catalyzing the first committed step in the l-lysine biosynthetic pathway, were cloned and expressed as N-oligoHistagged fusion proteins in Escherichia coli. The purified gene products revealed HCS activity, i.e. catalyzed the condensation of α-ketoglutarate with acetyl-coenzyme A to yield homocitrate. The recombinant enzymes were purified to homogeneity and characterized for their physical properties and substrate specificities. As determined by size-exclusion chromatography (SEC) and native page electrophoresis, both isoenzymes adopt multiple quaternary structures, with the homotetrameric one being the most abundant. The KM (acetyl-CoA)=0.8±0.15mM and KM (α-ketoglutarate)=0.113±0.02mM for His6CaLys21p and KM (acetyl-CoA)=0.48±0.09mM and KM (α-ketoglutarate)=0.152±0.03mM values for His6CaLys22p were determined. Both enzyme versions were inhibited by l-Lys, i.e. the end product of the α-aminoadipate pathway but Lys22p was more sensitive than Lys21p, with Ki (L-Lys)=128±8μM for His6CaLys21p and Ki (L-Lys)=4.37±0.68μM for His6CaLys22p. The isoforms of C. albicans HCS exhibited differential sensitivity to several l-Lys analogues. Most notably, dl-α-difluoromethyllysine strongly inhibited His6CaLys22p (IC50 32±3μM) but was not inhibitory at all towards His6CaLys21p. Differential sensitivity of recombinant C. albicans Δlys21/LYS22, LYS21/Δlys22 and Δlys21/Δlys22 mutant strains to lysine analog, 2-aminoethyl-l-cysteine and biochemical properties of homocitrate synthase isoforms suggest different roles of two HCS isoenzymes in α-aminoadipate pathway.