Wild‐Type and Mutant Forms of Homoisocitric Dehydrogenase in the Yeast Saccharomycopsis lipolytica

Abstract

Homoisocitric dehydrogenase (EC 1 .I .1 .I 55) has been purified 525-fold from the yeast Succharomycopsis lipoljtica with a yield of 25 ", . The preparation was judged to be homogeneous by electrophoresis under denaturing and non-denaturing conditions and by isoelectric focusing; it consisted of a single protein with molecular weight of 48000. In the presence of homoisocitric acid, a higher molecular weight was observed, suggesting a dimeric structure for the native enzyme.Complementing mutants devoid of homoisocitric dehydrogenase activity mapped at two closely linked loci (/w9 and /w10). Lysl0 mutants displayed NAD-reducing activity, whereas lys9 mutants retained some carboxylating activity.Our results are best explained by the assumption that the active enzyme is a dimer of identical subunits involved in successive dehydrogenation and decarboxylation steps.

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

“…Slow response of the enzyme, in terms of kinetic characteristics, with a rapid change of substrate concentration is probably related to the conformational change as the rate-limiting step in the enzymatic reaction. The same phenomenon was reported previously for other hysteretic enzymes (Frieden, 1970(Frieden, , 1979, including HIcDH of Saccharomycopsis lypolytica (Gaillardin et al, 1982). Data accumulated so far for HIcDHs from different sources indicated that the enzyme is activated by K + , Mn 2+ , or Mg 2+ ions (Gaillardin et al, 1982;Miyazaki, 2005a, b;Yamamoto et al, 2007).…”

“…Gel filtration analysis of HIcDH from S. lipolytica indicated that obtained results depend on enzyme concentration and the presence/absence of homoisocitrate. High protein concentration or the presence of the substrate stimulates formation of a dimeric form, whereas in the diluted solution and/or in the absence of the substrate, the enzyme exists as monomer (Gaillardin et al ., ). A similar phenomenon was found in our studies for CaHIcDH.…”

“…Slow response of the enzyme, in terms of kinetic characteristics, with a rapid change of substrate concentration is probably related to the conformational change as the rate‐limiting step in the enzymatic reaction. The same phenomenon was reported previously for other hysteretic enzymes (Frieden, , ), including HIcDH of Saccharomycopsis lypolytica (Gaillardin et al ., ).…”

“…a). These properties are similar to those reported for the enzyme from S. cerevisiae (Lin et al ., ) or S. lipolytica (Gaillardin et al ., ). On the other hand, HIcDH from D. radiodurans was completely inactive in the absence of monovalent cations (Miyazaki, , b).…”

“…Data accumulated so far for HIcDHs from different sources indicated that the enzyme is activated by K + , Mn 2+ ,or Mg 2+ ions (Gaillardin et al ., ; Miyazaki, , b; Yamamoto et al ., ). In our hands, His 6 CaHIcDH was slightly active in the absence of K + (0.4 U mg −1 ) but enzyme activity progressively increased upon addition of K + ions, to reach 1.5 U mg −1 in the presence of 10‐mM KCl, whereas addition of Na + had no effect on enzyme activity (Fig.…”