Abstract
Threonine synthase (TS), which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the γ-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at Cβ to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k(cat) and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, α-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the Cβ of the PLP-α-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the Cβ of the analog. These results indicated that the phosphate ion released from OPHS by γ-elimination acts as the base catalyst for the addition of water at Cβ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/deprotonation at Cγ.
Highlights
Hydratase, tryptophan synthase, and cysteine synthase, constitutes the -family of pyridoxal 5-phosphate (PLP) enzymes (2, 3)
Based on the crystal structure of the T. thermophilus HB8 Threonine synthase (TS) complexed with AP5, we pointed out the possibility that the phosphate released during 4 3 5 may reside at the active site and act as a base catalyst for the protonation/deprotonation at C␥ (8)
Catalytic Reaction of TS with OPHS and L-Vinylglycine— TS catalyzes the formation of L-threonine from OPHS as a physiological reaction (1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 1 in Scheme 1)
Summary
Materials—The recombinant TS was isolated and purified as previously described (8). All other chemicals were of the highest grade commercially available. (E)-4-(3-hydroxy-2methyl-5-(phosphonooxymethyl)pyridin-4-yl)-2-oxobut-3enoic acid (10) was synthesized according to the method of Schnackerz et al (11). Enzymatic Formation of L-Threonine—TS was incubated with various concentrations of OPHS in a final volume of 100 l of 50 mM PIPES, 100 mM KCl, 0.1 mM EDTA, pH 8.0. Equal volumes (75 l each) of solution A (28 M enzyme and 100 mM phosphate or sulfate) and solution B (various concentrations of L-threonine) were mixed. The spectral data were analyzed by ProKineticist II (Version 1.9, Applied Photophysics) to obtain the spectra of the reaction intermediates and the kinetic parameters. Crystallization, Data Collection, and Refinement—The apoenzyme of TS was crystallized by the microdialysis method. The model of 10 was built using MOE Version 2007.09 (Chemical Computing Group), and the topology and parameter files for refinement in CNS were generated with XPLO2D in the X-UTIL package (18). The coordinates for the apoenzyme and TS complexed with 10 have been deposited in the PDB with accession codes 3AEY and 3AEX, respectively
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