Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7

J Biol Chem. 2005 Mar 11;280(10):9698-705. doi: 10.1074/jbc.M411211200. Epub 2004 Dec 14.

Abstract

L-rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms. Glucose-1-phosphate thymidylyltransferase (RmlA, EC 2.7.7.24) catalyzes the first reaction of the four-step pathway of L-rhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate. Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain 7. In this study, we report the heterologous expression of the largest homologue (a 401 residue-long ST0452 protein) and characterization of its thermostable activity. RmlA enzymatic activity of this protein was detected from 65 to 100 degrees C, with a half-life of 60 min at 95 degrees C and 180 min at 80 degrees C. Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein. Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing alpha-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate but not alpha-D-glucosamine-1-phosphate. However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP. Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities. This is the first report of a thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Base Sequence
  • Cloning, Molecular
  • Conserved Sequence
  • DNA Primers
  • Enzyme Stability
  • Hot Temperature
  • Molecular Sequence Data
  • Nucleotidyltransferases / chemistry
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism*
  • Open Reading Frames
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Deletion
  • Sequence Homology, Amino Acid
  • Sulfolobus / enzymology*
  • Thermodynamics

Substances

  • Archaeal Proteins
  • DNA Primers
  • Recombinant Proteins
  • Nucleotidyltransferases