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 journalJournal articleResearchpeer-review

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, [k(cat)/K-m, NAD]/[k(cat)/K-m, NADP](+) 10.9, where K-m, NAD is the K-m for NAD(+) and K-m, NADP is the K-m 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, [k(cat)/K-m, NAD]/[k(cat)/K-m, 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
JournalAPPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume78
Issue number9
Pages (from-to)3079-3086
ISSN0099-2240
DOIs
Publication statusPublished - 2012
Externally publishedYes

Keywords

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Coenzymes
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • NAD
  • NADP
  • Pentoses
  • Protein Binding
  • Ribitol
  • Sequence Homology, Amino Acid
  • Sugar Alcohol Dehydrogenases
  • Zymomonas
  • 0U46U6E8UK NAD
  • 488-81-3 Ribitol
  • 53-59-8 NADP
  • 5556-48-9 ribulose
  • EC 1.1.- Sugar Alcohol Dehydrogenases
  • EC 1.1.1.56 ribitol 2-dehydrogenase
  • Molecular dynamics
  • Amino acids
  • aldehyde reductase
  • nicotinamide adenine dinucleotide
  • nicotinamide adenine dinucleotide phosphate
  • pentose
  • ribitol
  • ribitol 2 dehydrogenase
  • ribitol 2-dehydrogenase
  • ribulose
  • amino acid
  • bacterium
  • calorimetry
  • chemical binding
  • enzyme activity
  • homology
  • hydrophobicity
  • molecular analysis
  • amino acid sequence
  • amino acid substitution
  • article
  • coenzyme
  • enzymology
  • genetics
  • kinetics
  • metabolism
  • molecular genetics
  • protein binding
  • sequence homology
  • site directed mutagenesis
  • Zymomonas mobilis
  • Amino acid residues
  • Charged amino acids
  • Cofactor specificity
  • Cofactors
  • Higher efficiency
  • Isothermal titration calorimetry
  • Molecular determinants
  • Non-polar
  • Polar amino acids
  • Positively charged
  • Site directed mutagenesis
  • Substrate-binding
  • Wild types
  • catalytic efficiency
  • cofactor specificity
  • Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Endospore-forming Gram-Positives [07810] Bacillus megaterium species
  • Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Facultatively Anaerobic Gram-Negative Rods [06700] Zymomonas mobilis species
  • arginine 7200-25-1
  • asparagine 3130-87-8
  • D-ribulose 488-84-6
  • glucose-1-dehydrogenase EC 1.1.1.47
  • glutamic acid 617-65-2
  • histidine 4998-57-6
  • hydrophobic nonpolar amino acid
  • hydrophobic polar amino acid
  • lysine 70-54-2
  • NAD 53-84-9
  • NAD(P) 53-59-8
  • NAD(P)H 53-57-6
  • ribitol 488-81-3
  • ribitol dehydrogenase 9014-23-7 EC 1.1.1.56 catalyst
  • serine 302-84-1
  • 04500, Mathematical biology and statistical methods
  • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
  • 10064, Biochemistry studies - Proteins, peptides and amino acids
  • 10515, Biophysics - Biocybernetics
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 31000, Physiology and biochemistry of bacteria
  • Biochemistry and Molecular Biophysics
  • Computational Biology
  • homology modeling mathematical and computer techniques
  • isothermal titration calorimetry laboratory techniques
  • molecular dynamics simulation mathematical and computer techniques
  • site-directed mutagenesis laboratory techniques, genetic techniques
  • Enzymology
  • Methods and Techniques
  • Models and Simulations
  • BIOTECHNOLOGY
  • MICROBIOLOGY
  • 2,5-DIKETO-D-GLUCONIC ACID REDUCTASE
  • NADP-DEPENDENT ENZYMES
  • COENZYME SPECIFICITY
  • PHOSPHITE DEHYDROGENASE
  • GLUCOSE-DEHYDROGENASE
  • CARBONYL REDUCTASE
  • BINDING SITE
  • SUBSTITUTION
  • OXIDOREDUCTASE
  • EVOLUTION
  • DEHYDROGENASES
  • ZYMOMONAS
  • ZYMOMONAS MOBILIS
  • Biotechnology
  • Enzyme systems

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