Homoisocitrate dehydrogenase fromCandida albicans: properties, inhibition, and targeting by an antifungal pro-drug

Abstract

The LYS12 gene from Candida albicans, coding for homoisocitrate dehydrogenase was cloned and expressed as a His-tagged protein in Escherichia 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 CaHIcDH 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 CaHIcDH may exist as a monomer and as a tetramer and the latter form is favored by homoisocitrate binding. CaHIcDH is an hysteretic enzyme. The K(M) values of the purified His-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 CaHIcDH 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.

Inhibitors of amino acids biosynthesis as antifungal agents

“…The double null lys21Δ/lys22Δ mutant lacking homocitrate synthase activity exhibited lysine auxotrophy in minimal media but its virulence in vivo in the model of disseminated murine candidiasis appeared identical to that of the mother, wild-type strain (Kur et al 2010 ). Very similar phenotype was reported for the homoisocitrate dehydrogenase-deficient C. albicans mutant (Gabriel et al 2013 ). Interestingly, the same phenomenon was demonstrated for Cryptococcus neoformans cells auxotrophic for l -methionine due to the targeted disruption of homoserine transacetylase, in the mouse inhalation model (Nazi et al 2007 ).…”

“…Kinetic analysis showed that the compound was a noncompetitive inhibitor of this enzyme with respect to NAD + and competitive with respect to homoisocitrate, with K i = 2.91 mM. Comparing with other described compounds it can be classified as a weak inhibitor (Gabriel et al 2013 ). Antifungal in vitro activity of ( 2R,3S )-3-(p-carboxybenzyl)malate and its trimethyl ester was determined against some human pathogenic fungi from the Candida genus and S. cerevisiae .…”

‘Acridines’ as New Horizons in Antifungal Treatment

“…The second is to identify new targets specific for fungal cell and design effective inhibitors. As far as new targets are concerned, enzymes important for the biosynthesis of fungal proteins, essential amino acids or DNA are widely analyzed [3][4][5][6][7]. Moreover, the development of resistance, often multidrug resistance (MDR), is an emerging problem.…”

Consequences of lysine auxotrophy for Candida albicans adherence and biofilm formation

“…The potential of AAP enzymes as targets for antifungal chemotherapy has inspired the design and synthesis of structural analogs of AAP intermediates, which have been tested for antifungal activity and/or inhibition of the AAP enzymes. Some of these compounds had showed moderate inhibition of fungal growth that could be partially restored by the presence of L-lysine in the growth medium (Palmer et al, 2004;Gabriel et al, 2013).…”