Dihydrodipicolinate synthase (DHDPS, EC 18.104.22.168) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of ( S )-aspartate-β-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5 Å, revealing a tetrameric molecule comprised of four identical (β/α) 8 -barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35 Å resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.
Journal of Molecular Biology – Elsevier
Published: Apr 23, 2004
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