The PduX enzyme of Salmonella enterica is an L-threonine kinase used for coenzyme B12 synthesis.
Here, the PduX enzyme of Salmonella enterica is shown to be an L-threonine kinase used for the de novo synthesis of coenzyme B(12) and the assimilation of cobyric acid (Cby). PduX with a C-terminal His tag (PduX-His(6)) was produced at high levels in Escherichia coli, purified by nickel affinity chromatography, and partially characterized. (31)P NMR spectroscopy established that purified PduX-His(6) catalyzed the conversion of l-threonine and ATP to L-threonine-O-3-phosphate and ADP. Enzyme assays showed that ATP was the preferred substrate compared with GTP, CTP, or UTP. PduX displayed Michaelis-Menten kinetics with respect to both ATP and l-threonine and nonlinear regression was used to determine the following kinetic constants: V(max) = 62.1 +/- 3.6 nmol min(-1) mg of protein(-1); K(m)(, ATP) = 54.7 +/- 5.7 microm and K(m)(,Thr) = 146.1 +/- 8.4 microm. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B(12) de novo and from Cby, but not from cobinamide, which was the expected phenotype for an L-threonine kinase mutant. The defect in Cby assimilation was corrected by ectopic expression of pduX or by supplementation of growth medium with L-threonine-O-3-phosphate, providing further support that PduX is an L-threonine kinase. In addition, a bioassay showed that a pduX mutant was impaired for the de novo synthesis of coenzyme B(12) as expected. Collectively, the genetic and biochemical studies presented here show that PduX is an L-threonine kinase used for AdoCbl synthesis. To our knowledge, PduX is the first enzyme shown to phosphorylate free L-threonine and the first L-threonine kinase shown to function in coenzyme B(12) synthesis.
Fan
Chenguang
C
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Bobik
Thomas A
TA
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D002846:Chromatography, Affinity / Q000379:methods; D003038:Cobamides / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008956:Models, Chemical; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D019779:Salmonella enterica / Q000201:enzymology*; D013912:Threonine / Q000737:chemistry
D001426:Bacterial Proteins; D003038:Cobamides; D013912:Threonine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; C000913:cobamamide