K-068. The PduX Enzyme of Salmonella enterica Is a Threonine Kinase Used for Coenzyme B12 Synthesis

C. Fan, T. A. Bobik;
Iowa State Univ., Ames, IA.

Prior studies indicated that threonine-phosphate is an intermediate in the de novo synthesis of coenzyme B12. Bioinformatic studies showed that last gene in the pdu operon (pduX) has homology to GHMP kinases and that a number of pduX homologues are located proximal to genes for AdoCbl biosynthesis. This suggested the pduX gene might encode a threonine kinase. Here biochemical and genetic studies are performed that show the PduX enzyme of Salmonella enterica is a threonine kinase required for the synthesis of coenzyme B12. PduX with a C-terminal his tag (PduX-His6) was produced at high levels in Escherichia coli, purified using nickel-affinity chromatography, and partially characterized. 31P NMR spectroscopy showed that purified PduX-His6 catalyzed the conversion of L-threonine and ATP to L-threonine-O-3-phosphate and ADP. ATP was the preferred substrate compared to GTP, CTP or UTP. Initial rate studies showed that PduX displayed Michaelis-Menten kinetics with ATP and L-threonine with the following kinetic constants: Vmax = 62.13 ± 3.56 nmol min-1 mg-1; KmATP = 54.69 ± 5.66 μM, and KmThr = 146.06 ± 8.42 μM. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B12 de novo and from cobyric acid. Further growth studies showed that these B12 biosynthesis defects were corrected by supplementation of growth medium with L-threonine-phosphate indicating that PduX functions in the synthesis of this compound. Thus, genetic studies provided compelling evidence that PduX produces threonine phosphate for AdoCbl synthesis in vivo. To our knowledge, PduX is the first enzyme shown to phosphorylate free threonine and first threonine kinase shown to function in coenzyme B12 synthesis.