Characterization of recombinant homocitrate synthase from Candida albicans

Iwona Gabriel; Sławomir Milewski

doi:10.1016/j.pep.2015.09.005

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 NoligoHistagged 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 ± 0.15 mM and KM (α-ketoglutarate) = 0.113 ± 0.02 mM for His6CaLys21p and KM (acetyl-CoA) = 0.48 ± 0.09 mM and KM (α-ketoglutarate) = 0.152 ± 0.03 mM 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 ± 8 μM for His6CaLys21p and Ki (LLys) = 4.37 ± 0.68 μM 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 ± 3 μM) 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.

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Authors (2)

Iwona Gabriel dr hab. inż.
Sławomir Milewski prof. dr hab. inż.

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Category:: Articles
Type:: artykuł w czasopiśmie wyróżnionym w JCR
Published in:: PROTEIN EXPRESSION AND PURIFICATION no. 125, pages 7 - 18,
ISSN: 1046-5928
Language:: English
Publication year:: 2016
Bibliographic description:: Gabriel I., Milewski S.: Characterization of recombinant homocitrate synthase from Candida albicans// PROTEIN EXPRESSION AND PURIFICATION. -Vol. 125, (2016), s.7-18
DOI:: Digital Object Identifier (open in new tab) 10.1016/j.pep.2015.09.005
Verified by:: Gdańsk University of Technology