The PduX enzyme of Salmonella enterica is an L-threonine kinase used for coenzyme B12 synthesis

Abstract

Here, the PduX enzyme of Salmonella enterica is shown to be an L-threonine kinase used for the de novo synthesis of coenzyme B12 and the assimilation of cobyric acid (Cby). PduX with a C-terminal His tag (PduX-His6) was produced at high levels in Escherichia coli, purified by nickel affinity chromatography, and partially characterized. 31P NMR spectroscopy established that purified PduX-His6 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: Vmax = 62.1 ± 3.6 nmol min-1 mg of protein-1; Km,ATP = 54.7 ± 5.7 μM and K m,Thr = 146.1 ± 8.4 μM. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B12 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 B12 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 B12 synthesis. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.