Cloning and characterization of a ribitol dehydrogenase from Zymomonas mobilis

Hee-Jung Moon, Manish Kumar Tiwari, Marimuthu Jeya, Jung-Kul Lee

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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 ∼28 kDa by sodium dodecyl sulfate-polyacrylamide gel and ∼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°C, respectively. Unlike previously characterized RDHs, Z. mobilis RDH (ZmRDH) showed an unusual dual coenzyme specificity, with a kcat of
4.83 s−1 for NADH (kcat/Km=27.3 s−1mM−1) and kcat of 2.79 s−1 for NADPH (kcat/Km=10.8 s−1mM−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.
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume87
Pages (from-to)205–214
ISSN0175-7598
DOIs
Publication statusPublished - 2010
Externally publishedYes

Keywords

  • Characterization
  • Coenzyme specificity
  • Zymomonas mobilis
  • Ribitol dehydrogenase
  • Ribulose

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