Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase

Hee Jung Moon, Manish Kumar Tiwari, Ranjitha Singh, Yun Chan Kang, Jung Kul Lee

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to D-ribulose and concomitantly reduces NAD(P)+ to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+ (S156D, [kcat/Km, NAD]/[kcat/Km, NADP]=10.9, where Km, NAD is the Km for NAD+ and Km, NADP is the Km for NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+ as the cofactor (S156H, [kcat/Km, NAD]/[kcat/Km, NADP]=0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

Original languageEnglish
Pages (from-to)3079-3086
Number of pages8
JournalApplied and Environmental Microbiology
Volume78
Issue number9
DOIs
Publication statusPublished - 2012 May 1
Externally publishedYes

Fingerprint

ribitol 2-dehydrogenase
NAD
Oxidoreductases
amino acid
NADP
amino acids
Amino Acids
Zymomonas mobilis
screening
ribulose
molecular dynamics
site-directed mutagenesis
calorimetry
sequence alignment
Ribitol
Zymomonas
titration
homology
Calorimetry
Sequence Alignment

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

Cite this

Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase. / Moon, Hee Jung; Tiwari, Manish Kumar; Singh, Ranjitha; Kang, Yun Chan; Lee, Jung Kul.

In: Applied and Environmental Microbiology, Vol. 78, No. 9, 01.05.2012, p. 3079-3086.

Research output: Contribution to journalArticle

Moon, Hee Jung ; Tiwari, Manish Kumar ; Singh, Ranjitha ; Kang, Yun Chan ; Lee, Jung Kul. / Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase. In: Applied and Environmental Microbiology. 2012 ; Vol. 78, No. 9. pp. 3079-3086.
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