Kinetic and functional analysis of L-threonine kinase, the PduX enzyme of Salmonella enterica

J Biol Chem. 2009 Jul 24;284(30):20240-8. doi: 10.1074/jbc.M109.027425. Epub 2009 Jun 9.


The PduX enzyme of Salmonella enterica is an l-threonine kinase used for the de novo synthesis of coenzyme B(12) and the assimilation of cobyric acid. PduX with an N-terminal histidine tag (His(8)-PduX) was produced in Escherichiacoli and purified. The recombinant enzyme was soluble and active. Kinetic analysis indicated a steady-state Ordered Bi Bi complex mechanism in which ATP is the first substrate to bind. Based on a multiple sequence alignment of PduX homologues and other GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family members, 14 PduX variants having changes at 10 conserved serine/threonine and aspartate/glutamate sites were constructed by site-directed mutagenesis. Each variant was produced in E. coli and purified. Comparison of the circular dichroism spectra and kinetic properties of the PduX variants with those of the wild-type enzyme indicated that Glu-24 and Asp-135 are needed for proper folding, Ser-99 and Glu-132 are used for ATP binding, and Ser-253 and Ser-255 are critical to l-threonine binding whereas Ser-100 is essential to catalysis, but its precise role is uncertain. The studies reported here are the first to investigate the kinetic and catalytic mechanisms of l-threonine kinase from any organism.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism
  • Amino Acid Motifs
  • Circular Dichroism
  • Enzyme Inhibitors / metabolism
  • Enzyme Stability
  • Escherichia coli / genetics
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutant Proteins / analysis
  • Mutant Proteins / antagonists & inhibitors
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Protein Serine-Threonine Kinases / analysis
  • Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Protein Serine-Threonine Kinases / genetics*
  • Protein Serine-Threonine Kinases / metabolism*
  • Salmonella enterica / enzymology*
  • Sequence Alignment
  • Threonine / analogs & derivatives
  • Threonine / metabolism


  • Enzyme Inhibitors
  • Mutant Proteins
  • Threonine
  • Adenosine Triphosphate
  • Protein Serine-Threonine Kinases