Cloning and characterization of a ribitol dehydrogenase from Zymomonas mobilis.
Ribitol dehydrogenase (RDH) catalyzes the conversion of ribitol to D-ribulose. A novel RDH gene was cloned from Zymomonas mobilis subsp. mobilis ZM4 and overexpressed in Escherichia coli BL21(DE3). DNA sequence analysis revealed an open reading frame of 795 bp, capable of encoding a polypeptide of 266 amino acid residues with a calculated molecular mass of 28,426 Da. The gene was overexpressed in E. coli BL21(DE3) and the protein was purified as an active soluble form using glutathione S-transferase affinity chromatography. The molecular mass of the purified enzyme was estimated to be approximately 28 kDa by sodium dodecyl sulfate-polyacrylamide gel and approximately 58 KDa with gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 9.5 and 65 degrees C, respectively. Unlike previously characterized RDHs, Z. mobilis RDH (ZmRDH) showed an unusual dual coenzyme specificity, with a k(cat) of 4.83 s(-1) for NADH (k(cat)/K(m) = 27.3 s(-1) mM(-1)) and k(cat) of 2.79 s(-1) for NADPH (k(cat)/K(m) = 10.8 s(-1) mM(-1)). Homology modeling and docking studies of NAD+ and NADP+ into the active site of ZmRDH shed light on the dual coenzyme specificity of ZmRDH.
Moon
Hee-Jung
HJ
Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, South Korea 143-701.
Tiwari
Manish
M
Jeya
Marimuthu
M
Lee
Jung-Kul
JK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017355:Zymomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D013401:Sugar Alcohol Dehydrogenases; C022529:ribitol 2-dehydrogenase