Cloning, expression, and enzymatic characterization of isocitrate dehydrogenase from Helicobacter pylori.
Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate with NAD(P) as a cofactor in the tricarboxylic acid cycle. As a housekeeping protein in Helicobacter pylori, IDH was considered as a possible candidate for serological diagnostics and detection. Here, we identified a new icd gene encoding IDH from H. pylori strain SS1. The recombinant H. pylori isocitrate dehydrogenase (HpIDH) was cloned, expressed, and purified in E. coli system. The enzymatic characterization of HpIDH demonstrates its activity with k (cat) of 87 s(-1), K (m) of 124 microM and k (cat)/K (m) of 7 x 10(5) M(-1)s(-1) toward isocitrate, k (cat) of 80 s(-1), K (m) of 176 microM and k (cat)/K (m) of 4.5 x 10(5) M(-1)s(-1) toward NADP. The optimum pH of the enzyme activity is around 9.0, and the optimum temperature is around 50 degrees C. This current work is expected to help better understand the features of HpIDH and provide useful information for H. pylori serological diagnostics and detection.
Huang
Dongsheng
D
Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 310016 Hangzhou, China. dshuang1016@yahoo.cn.
Liu
Junwei
J
Shen
Guoliang
G
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000201:enzymology*; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D007521:Isocitrate Dehydrogenase