Elucidation of the role of Ser329 and the C-terminal region in the catalytic activity of Pseudomonas stutzeri L-rhamnose isomerase

Protein Eng Des Sel. 2010 Dec;23(12):919-27. doi: 10.1093/protein/gzq077. Epub 2010 Oct 25.


Pseudomonas stutzeri l-rhamnose isomerase (l-RhI) is capable of catalyzing the isomerization between various aldoses and ketoses, showing high catalytic activity with broad substrate-specificity compared with Escherichia coli l-RhI. In a previous study, the crystal structure of P. stutzeri l-RhI revealed an active site comparable with that of E. coli l-RhI and d-xylose isomerases (d-XIs) with structurally conserved amino acids, but also with a different residue seemingly responsible for the specificity of P. stutzeri l-RhI, though the residue itself does not interact with the bound substrate. This residue, Ser329, corresponds to Phe336 in E. coli l-RhI and Lys294 in Actinoplanes missouriensis d-XI. To elucidate the role of Ser329 in P. stutzeri l-RhI, we constructed mutants, S329F (E. coli l-RhI type), S329K (A. missouriensis d-XI type), S329L and S329A. Analyses of the catalytic activity and crystal structure of the mutants revealed a hydroxyl group of Ser329 to be crucial for catalytic activity via interaction with a water molecule. In addition, in complexes with substrate, the mutants S329F and S329L exhibited significant electron density in the C-terminal region not observed in the wild-type P. stutzeri l-RhI. The C-terminal region of P. stutzeri l-RhI has flexibility and shows a flip-flop movement at the inter-molecular surface of the dimeric form.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldose-Ketose Isomerases / chemistry*
  • Aldose-Ketose Isomerases / genetics
  • Aldose-Ketose Isomerases / metabolism*
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biocatalysis
  • Catalytic Domain
  • Escherichia coli / enzymology
  • Hexoses / chemistry
  • Hexoses / metabolism
  • Molecular Sequence Annotation
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation / genetics
  • Mutation / physiology
  • Protein Structure, Tertiary
  • Pseudomonas stutzeri / enzymology*
  • Pseudomonas stutzeri / genetics
  • Sequence Analysis, Protein
  • Sequence Homology, Amino Acid
  • Serine / genetics
  • Serine / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity
  • X-Ray Diffraction


  • Bacterial Proteins
  • Hexoses
  • Serine
  • Aldose-Ketose Isomerases
  • L-rhamnose isomerase