2d0d
Crystal Structure of a Meta-cleavage Product Hydrolase (CumD) A129V Mutant
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OverviewOverview
The meta-cleavage product hydrolase from Pseudomonas fluorescens IP01, (CumD) hydrolyzes 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl, HODA) in the cumene (isopropylbenzene) degradation pathway. To modulate, the substrate specificity and catalytic efficiency of CumD toward, substrates derived from monocyclic aromatic compounds, we constructed the, CumD mutants, A129V, I199V, and V227I, as well as four types of double and, triple mutants. Toward substrates with smaller side chains (e.g., 2-hydroxy-6-oxohepta-2,4-dienoate; 6-ethyl-HODA), the k(cat)/K(m) values, of the single mutants were 4.2-11 fold higher than that of the wild type, enzyme and 1.8-4.7 fold higher than that of the meta-cleavage product, hydrolase from Pseudomonas putida F1 (TodF). The A129V mutant showed the, highest k(cat)/K(m) value for 2-hydroxy-6-oxohepta-2,4-dienoate, (6-ethyl-HODA). The crystal structure of the A129V mutant was determined, at 1.65 A resolution, enabling location of the Ogamma atom of the Ser103, side chain. A chloride ion was bound to the oxyanion hole of the active, site, and mutant enzymes at the residues forming this site were also, examined. The k(cat) values of Ser34 mutants were decreased 2.9-65 fold, suggesting that the side chain of Ser34 supports catalysis by stabilizing, the anionic oxygen of the proposed intermediate state (gem-diolate). This, is the first crystal structure determination of CumD in an active form, with the Ser103 residue, one of the catalytically essential "triad", being, intact.
About this StructureAbout this Structure
2D0D is a Single protein structure of sequence from Pseudomonas fluorescens with and as ligands. Active as 2-hydroxymuconate-semialdehyde hydrolase, with EC number 3.7.1.9 Full crystallographic information is available from OCA.
ReferenceReference
Improving the catalytic efficiency of a meta-cleavage product hydrolase (CumD) from Pseudomonas fluorescens IP01., Jun SY, Fushinobu S, Nojiri H, Omori T, Shoun H, Wakagi T, Biochim Biophys Acta. 2006 Jul;1764(7):1159-66. Epub 2006 Jun 7. PMID:16844437
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