Abstract

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of (S)-aspartate-beta-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5 Angstrom, revealing a tetrameric molecule comprised of four identical (beta/alpha)(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 Angstrom 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. (C) 2004 Elsevier Ltd. All rights reserved

Published in

Journal of Molecular Biology
2004, Volume: 338, number: 2, pages: 329-339
Publisher: ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD

SLU Authors

• Valegård, Karin

  • Department of Molecular Biology, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Food Science


Publication identifier

DOI: https://doi.org/10.1016/j.jmb.2004.02.060

Permanent link to this page (URI)

https://res.slu.se/id/publ/6074