title: Enzymatic characterization and functional implication of two structurally different isocitrate dehydrogenases from Xylella fastidiosa.
abstract: Isocitrate dehydrogenase (IDH) is a key enzyme at the critical junction between the tricarboxylic acid cycle and the glyoxylate cycle. Most bacteria have only one IDH, while a few contain two IDH isozymes. The coexistence of two different type IDHs in one organism was little known. Xylella fastidiosa is a nutritionally fastidious plant pathogen that contains two structurally different IDHs, an NAD+-dependent homodimeric IDH (diXfIDH) and an NADP+-dependent monomeric IDH (monoXfIDH). Kinetic characterization showed that diXfIDH displayed 206-fold preferences for NAD+over NADP+, while monoXfIDH showed 13,800-fold preferences for NADP+over NAD+. The putative coenzyme crucial amino acids (Asp-268, Ile-269, and Ala-275 in diXfIDH, and Lys-589, His-590, and Arg-601 in monoXfIDH) were studied by site-directed mutagenesis. The coenzyme specificities of the three diXfIDH mutants (D268K, D268K/I269Y, and D268K/I269Y/A275V) were switched successfully from NAD+to NADP+. Meanwhile, the mutant monoXfIDHs (H590L/R601L and K589T/H590L/R601L) greatly reduced the affinity for NADP+, but failed to improve the ability to use NAD+and had similar affinity to NADP+and NAD+. The biochemical properties of diXfIDH and monoXfIDH were investigated in detail. This study gives a further insight into the determinants of the coenzyme specificity in both monomeric and dimeric forms of IDHs.
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Peipei
authors.initials: P
authors.lastname: Lv
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, Anhui, People's Republic of China.|Department of Biochemistry and Molecular Biology, Bengbu Medical College, Bengbu, People's Republic of China.
authors.forename: Wanggang
authors.initials: W
authors.lastname: Tang
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
authors.affiliation: The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010802:Phylogeny; D010944:Plants / Q000382:microbiology; D011487:Protein Conformation; D055503:Protein Multimerization; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D044167:Xylella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D009243:NAD; D009249:NADP; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)
keywords: Xylella fastidiosa; biochemical properties; coenzyme specificity; isocitrate dehydrogenase; site-directed mutagenesis