Abstract
Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open alpha/beta architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.
Highlights
Threonine synthase (TS, EC 4.2.99.2)1 is a pyridoxal 5Јphosphate (PLP)-dependent enzyme that catalyzes the ultimate step in threonine biosynthesis, the pyridoxal 5-phosphate (PLP)-dependent ,␥
TS from higher plants occurs in solution as homodimers of about 110 kDa, and detailed studies with the A. thaliana enzyme indicated that the extended N terminus is responsible for allosteric activation [7, 16]
TS from the fungal class are unique among PLP-dependent enzymes, because they fulfill their physiological function in monomeric state [8, 10]
Summary
The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 Å resolution using multiwavelength anomalous diffraction. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. The substrate of TS, OPHS, is the branching point for threonine and methionine biosynthesis. Flux coordination between both synthetic pathways is accomplished by allosteric activation of plant threonine synthase. As a first step toward structure-based drug design, we report here the crystal structure of TS from Saccharomyces cerevisiae (yTS) at 2.7 Å resolution This is the first structure of a PLP enzyme that is active as monomer. Comparison with related enzymes and modeling of the physiological enzyme-substrate complex give new insight into the reaction mechanism of threonine synthases
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