Abstract

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.

Highlights

  • Cobyric acid (Cby) under both aerobic and anaerobic conditions (3, 5)

  • These findings predicted that an L-threonine (L-Thr) kinase would be required for the de novo synthesis of AdoCbl and MeCbl, and the assimilation of cobyric acid (Cby)

  • Production of MeCbl Sufficient to Support Methionine Biosynthesis Is Independent of PduX—S. enterica metE mutants require MeCbl for methionine biosynthesis; L-Thr kinase should be required for growth of metE strains on minimal medium supplemented with Cby (Fig. 1) (14)

Read more

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Reagents—Antibiotics, Cbi, L-Thr-P, (R)-1amino-2-propanol (AP), nucleoside triphosphates, and nucleoside diphosphates were purchased from Sigma. Restriction enzymes and T4 DNA ligase were from New England Biolabs, 11322 JOURNAL OF BIOLOGICAL CHEMISTRY

Species and strain
This study This study This study This study
DISCUSSION
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call