2d0d

Crystal Structure of a Meta-cleavage Product Hydrolase (CumD) A129V Mutant

spinqualitylabelsall models 2d0d, resolution 1.65Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 2d0d is a 1 chain structure with sequence from "bacillus_fluorescens_liquefaciens"_flugge_1886 "bacillus fluorescens liquefaciens" flugge 1886. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: CL, PO4 Related: 1iup Gene: CUMD ("Bacillus fluorescens liquefaciens" Flugge 1886) Activity: 2-hydroxymuconate-6-semialdehyde hydrolase, with EC number 3.7.1.9 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed 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. 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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