Open AccessHomoisocitrate dehydrogenase from C andida albicans : properties, inhibition, and targeting by an antifungal pro‐drug
Author(s) -
Gabriel Iwona,
Vetter Natasha D.,
Palmer David R.J.,
Milewska Maria J.,
Wojciechowski Marek,
Milewski Sławomir
Publication year - 2013
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/1567-1364.12014
Subject(s) - enzyme , molecular mass , biology , biochemistry , tetramer , candida albicans , escherichia coli , enzyme assay , recombinant dna , enzyme kinetics , microbiology and biotechnology , dehydrogenase , active site , gene
The LYS12 gene from C andida albicans , coding for homoisocitrate dehydrogenase was cloned and expressed as a H is‐tagged protein in E scherichia coli . The purified gene product catalyzes the Mg 2+ ‐ and K + ‐dependent oxidative decarboxylation of homoisocitrate to α‐ketoadipate. The recombinant enzyme demonstrates strict specificity for homoisocitrate. SDS ‐ PAGE of C a HI c DH revealed its molecular mass of 42.6 ± 1 kDa, whereas in size‐exclusion chromatography, the enzyme eluted in a single peak corresponding to a molecular mass of 158 ± 3 kDa. Native electrophoresis showed that C a HI c DH may exist as a monomer and as a tetramer and the latter form is favored by homoisocitrate binding. C a HI c DH is an hysteretic enzyme. The K M values of the purified H is‐tagged enzyme for NAD + and homoisocitrate were 1.09 mM and 73.7 μM, respectively, and k cat was 0.38 s −1 . Kinetic parameters determined for the wild‐type C a HI c DH were very similar. The enzyme activity was inhibited by ( 2R,3S )‐3‐( p ‐carboxybenzyl)malate ( CBMA ), with IC 50 = 3.78 mM. CBMA demonstrated some moderate antifungal activity in minimal media that could be enhanced upon conversion of the enzyme inhibitor into its trimethyl ester derivative ( TMCBMA ). TMCBMA is the first reported antifungal for which an enzyme of the AAP was identified as a molecular target.