Kinetic and functional analysis of L-threonine kinase, the PduX enzyme of Salmonella enterica

Abstract

The PduX enzyme of Salmonella enterica is an l-threonine kinase used for the de novo synthesis of coenzyme B(12) and the assimilation of cobyric acid. PduX with an N-terminal histidine tag (His(8)-PduX) was produced in Escherichiacoli and purified. The recombinant enzyme was soluble and active. Kinetic analysis indicated a steady-state Ordered Bi Bi complex mechanism in which ATP is the first substrate to bind. Based on a multiple sequence alignment of PduX homologues and other GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family members, 14 PduX variants having changes at 10 conserved serine/threonine and aspartate/glutamate sites were constructed by site-directed mutagenesis. Each variant was produced in E. coli and purified. Comparison of the circular dichroism spectra and kinetic properties of the PduX variants with those of the wild-type enzyme indicated that Glu-24 and Asp-135 are needed for proper folding, Ser-99 and Glu-132 are used for ATP binding, and Ser-253 and Ser-255 are critical to l-threonine binding whereas Ser-100 is essential to catalysis, but its precise role is uncertain. The studies reported here are the first to investigate the kinetic and catalytic mechanisms of l-threonine kinase from any organism.

FIGURE 1.

Roles of PduX in B₁₂ synthesis in S. enterica. PduX is required for both the de novo synthesis of AdoCbl and methylcobalamin (MeCbl) and the assimilation of cobyric acid. AdoCbl is required for growth of S. enterica on 1,2-PD and ethanolamine as well for the activity of the MetH methionine synthase that converts homocysteine to methionine. Btu, B₁₂ uptake system; Cbi, cobinamide; AdoCbi, adenosylcobinamide; AdoCby, adenosylcobyric acid; AdoCbi-P, adenosylcobinamide phosphate; AP-P, (R)-1-amino-2-propanol-O-2-phosphate.

FIGURE 2.

Initial rate study of the PduX reaction. Double reciprocal plots of rate versus substrate concentration with one substrate varied and the other held at fixed levels are shown. The reaction conditions were as follows: 10 nm PduX enzyme and ATP and l-Thr concentrations as indicated. Standard assay mixtures contained 15 mm HEPES (pH 7.4), 20 mm NaCl, and 10 mm MgCl₂ in a total volume of 0.1 ml. The values and error bars shown are based on three replicates.

FIGURE 3.

Kinetic analysis of dead-end inhibition by the ATP analog AMP-PNP. A, AMP-PNP is a noncompetitive inhibitor of l-Thr in the PduX reaction. ATP concentration was held at 2 mm. B, AMP-PNP is a competitive inhibitor of ATP in the PduX reaction. l-Thr concentration was held at 5 mm. The reaction conditions were the same as those for the standard assay. The values and error bars shown are based on three replicates.

FIGURE 4.

Kinetic analysis of dead-end inhibition by the l-threonine analog l-valine. A, l-Val is a competitive inhibitor of l-Thr in the PduX reaction. ATP concentration was held at 100 μm. B, l-Val is an uncompetitive inhibitor of ATP in the PduX reaction. l-Thr concentration was held at 300 μm. The reaction conditions were the same as those for the standard assay. The values and error bars shown are based on three replicates.

FIGURE 5.

Kinetic analysis of product inhibition by l-Thr-P. A, l-Thr-P is a noncompetitive inhibitor of l-Thr in the PduX reaction. ATP concentration was held at 2 mm. B, l-Thr-P is a noncompetitive inhibitor of ATP in the PduX reaction. l-Thr concentration was held at 5 mm. The reaction conditions were the same as those for the standard assay. The values and error bars shown are based on three replicates.

FIGURE 6.

Kinetic mechanism of PduX l-Thr kinase. The kinetic mechanism of PduX is an ordered ternary complex mechanism with ATP binding first.

FIGURE 7.

Motifs found in a multiple sequence alignment of PduX and homologues. A multiple sequence alignment identified four conserved motifs among PduX homologues. The corresponding motif in the GHMP family is indicated. Motif four is characteristic of PduX. Motif I is only weakly conserved. Deduced amino acid sequences of 18 PduX homologues were aligned using the program ClustalX. Residue numbering corresponds to the PduX enzyme of Salmonella. Invariant residues are marked by stars and highlighted with black boxes and white font. The abbreviations for organisms in sequence after S. enterica are Citrobacter freundii, Klebsiella pneumoniae, Photorhabdus luminescens, Yersinia enterocolitica, Listeria welshimeri, Alkaliphilus metalliredigens, Heliobacterium modesticaldum, Thermosinus carboxydivorans, Moorella thermoacetica, Carboxydothermus hydrogenoformans, Natranaerobius thermophilus, Desulfitobacterium hafniense, Burkholderia pseudomallei, Actinoplanes friuliensis, Streptococcus sanguinis, Bacillus thuringiensis, and Clostridium botulinum.

FIGURE 8.

Stability of PduX variants. To estimate the stability of the PduX variants, unfolding by urea was followed by fluorescence spectroscopy. PduX variants were dissolved in 10 mm HEPES (pH 7.0) and 100 mm NaCl. The urea concentration was increased as indicated, and fluorescence intensity was measured. Data were taken at 25 °C, and ΔG_UN⁰ was calculated as described under “Experimental Procedures.” With the exception of E24A and D135A, most of the mutants exhibited unfolding curves with ΔG_UN⁰ values similar to that of wild type (Table 1). Solid squares, wild type; open diamonds, E24A; solid triangles, D135A.

FIGURE 9.

In vivo activities of PduX variants. Cells were grown in minimal 1,2-PD medium (described under “Experimental Procedures”) supplemented with 50 nm cobyric acid. PduX is required for growth under these conditions because it is essential for the conversion of Cby to AdoCbl, a required cofactor for 1,2-PD degradation by S. enterica. Three representative growth patterns are shown in the figure. The PduX variants displaying these patterns are described in the text. Solid diamond, wild type; open triangle, S253A; solid square, S100A.

FIGURE 10.

Proposed transition state for the PduX reaction. In this mechanism, “X” is either a catalytic base or a hydrogen bond acceptor that would help stabilize negative charge on the substrate hydroxyl increasing its nucleophilicity. The “Y” represents an acidic group on the enzyme that would stabilize developing negative charge and donate a proton to form the ADP dianion as the leaving group. In addition, the developing negative charge on the β-phosphate would likely be further stabilized by hydrogen bonding and coordination to divalent magnesium ion, which is not shown in the figure. Partial charges (δ) on groups X and Y were omitted because the exact nature of these groups is unknown.