title: NADP(+)-specific isocitrate dehydrogenase from oleaginous yeast Yarrowia lipolytica CLIB122: biochemical characterization and coenzyme sites evaluation.
abstract: NADP(+)-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg(2+) was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP(+) and no NAD-dependent activity could be detected. The K m values displayed for NADP(+) and isocitrate were 59 and 31 μM (Mg(2+)), 120 μM and 58 μM (Mn(2+)), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K m values for NADP(+) of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD(+)-dependent activity was detected for R322D mutant and the K m and k cat values for NAD(+) were 47,000 μM and 0.38 s(-1), respectively. Although the R322D mutant showed low activity with NAD(+), it revealed the feasibility of engineering an eukaryotic IDP to a NAD(+)-dependent one.
authors.affiliation: Institute of Molecular Biology and Biotechnology and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu 241000, Anhui, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Li
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Yadong
authors.initials: Y
authors.lastname: Ge
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.forename: Abdulla
authors.initials: A
authors.lastname: Abbas
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D021381:Protein Transport; D017421:Sequence Analysis; D013379:Substrate Specificity; D013696:Temperature; D025062:Yarrowia / Q000166:cytology / Q000201:enzymology*
chemical_list: D003067:Coenzymes; D008670:Metals; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)