Homoisocitrate dehydrogenase from C andida albicans: properties, inhibition, and targeting by an antifungal pro-drug

• Published in: FEMS yeast research 2013-03, Vol.13 (2), p.143-155
• Main Authors: Gabriel, Iwona, Vetter, Natasha D, Palmer, David RJ, Milewska, Maria J, Wojciechowski, Marek, Milewski, Sawomir
• Format: Article
• Language: English
• Subjects: Antifungal activity; Escherichia coli
• ISSN: 1567-1356; 1567-1364
• DOI: 10.1111/1567-1364.12014

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 super(2+) - and K super(+) -dependent oxidative decarboxylation of homoisocitrate to [alpha]-ketoadipate. The recombinant enzyme demonstrates strict specificity for homoisocitrate. SDS - PAGE of C a HI c DH revealed its molecular mass of 42.6 plus or minus 1 kDa, whereas in size-exclusion chromatography, the enzyme eluted in a single peak corresponding to a molecular mass of 158 plus or minus 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 sub(M) values of the purified H is-tagged enzyme for NAD super(+) and homoisocitrate were 1.09 mM and 73.7 mu M, respectively, and k sub(cat) was 0.38 s super(-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 sub(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.