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J. Biol. Chem., Vol. 283, Issue 17, 11322-11329, April 25, 2008

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The PduX Enzyme of Salmonella enterica Is an L-Threonine Kinase Used for Coenzyme B₁₂ Synthesis^*

From the Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011

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: V_max = 62.1 ± 3.6 nmol min^–1 mg of protein^–1; K_{m, 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.

Received for publication, January 11, 2008 , and in revised form, February 26, 2008.

^* This work was supported by Grant MCB0616008 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 515-294-4165; Fax: 515-294-0453; E-mail: bobik{at}iastate.edu.

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