title: Characterization of recombinant homocitrate synthase from Candida albicans.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 11/12 Narutowicza Str., 80-233 Gdansk, Poland. Electronic address: iwogabri@pg.gda.pl.
authors.forename: Iwona
authors.initials: I
authors.lastname: Gabriel
authors.affiliation: Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 11/12 Narutowicza Str., 80-233 Gdansk, Poland.
authors.forename: Sławomir
authors.initials: S
authors.lastname: Milewski
mesh_terms: D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000494:pharmacology; D008239:Lysine / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D007656:Ketoglutaric Acids; D008670:Metals; D011994:Recombinant Proteins; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases; D008239:Lysine
keywords: Candida albicans; Homocitrate synthase; Inhibition; α-Aminoadipate pathway