Site-directed mutagenesis of the active site region in the quinate/shikimate 5-dehydrogenase YdiB of Escherichia coli

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DOIResolve DOI: http://doi.org/10.1074/jbc.M412028200
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TypeArticle
Journal titleJournal Of Biological Chemistry
Volume280
Issue8
Pages71627169; # of pages: 8
Subject5'-Guanylic Acid; Escherichia coli; Mutagenesis; pha
AbstractYdiB and its paralog AroE are members of the quinate/shikimate 5-dehdrogenase family. Enzymes from this family function in the shikimate pathway that is essential for survival of microorganisms and plants and represent potential drug targets. Recent YdiB and AroE crystal structures revealed the presence of a NAD(P)-binding and a catalytic domain. We carried out site-directed mutagenesis of 8 putative active site residues in YdiB from Escherichia coli and analyzed structural and kinetic properties of the mutant enzymes. Our data indicate critical roles for an invariant lysine and aspartate residue in substrate binding and allowed us to differentiate between two previously proposed models for the binding of the substrate in the active site. Comparison of several YdiB and AroE structures led us to conclude that, upon cofactor binding and domain closure, the 2 identified binding residues are repositioned to bind to the substrate. Although the lysine residue contributes to some extent to the stabilization of the transition state, we did not identify any residue as catalytically essential. This indicates that catalysis does not operate through a general acid-base mechanism, as thought originally. Our improved understanding of the medically and agriculturally important quinate/shikimate 5-dehydrogenase family at the molecular level may prove useful in the development of novel herbicides and antimicrobial agents.
Publication date
LanguageEnglish
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedNo
NRC number46231
NPARC number3539417
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Record identifiere81eeca8-292d-4c13-a1e6-ecad18feb080
Record created2009-03-01
Record modified2016-05-09
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