2og1
Crystal Structure of BphD, a C-C hydrolase from Burkholderia xenovorans LB400
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OverviewOverview
Kinetic and structural analyses of, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from, Burkholderia xenovorans LB400 (BphD(LB400)) provide insight into the, catalytic mechanism of this unusual serine hydrolase. Single turnover, stopped-flow analysis at 25 degrees C showed that the enzyme rapidly, (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm), into a species with electronic absorption maxima at 473 and 492 nm. The, absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner, (1/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately k(cat)) with, simultaneous biphasic appearance (48 and 8 s(-1)) of an absorbance band at, 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic, acid (HPD). Increasing solution viscosity with glycerol slowed 1/tau(1), and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that, 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(3) represents, an intramolecular event. Product inhibition studies suggested that the, other product, benzoate, is released after HPD. Contrary to studies in a, related hydrolase, we found no evidence that ketonized HOPDA is partially, released prior to hydrolysis, and, therefore, postulate that the biphasic, kinetics reflect one of two mechanisms, pending assignment of E:S, (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C, variant, and S112C incubated with HOPDA were each determined to 1.6 A, resolution. The latter reveals interactions between conserved active site, residues and the dienoate moiety of the substrate. Most notably, the, catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo, substituent of HOPDA, consistent with a role in catalyzing ketonization., The data are more consistent with an acyl-enzyme mechanism than with the, formation of a gem-diol intermediate.
About this StructureAbout this Structure
2OG1 is a Single protein structure of sequence from Burkholderia xenovorans with SO4, GOL and EOH as ligands. Active as 2,6-dioxo-6-phenylhexa-3-enoate hydrolase, with EC number 3.7.1.8 Full crystallographic information is available from OCA.
ReferenceReference
Kinetic and structural insight into the mechanism of BphD, a C-C bond hydrolase from the biphenyl degradation pathway., Horsman GP, Ke J, Dai S, Seah SY, Bolin JT, Eltis LD, Biochemistry. 2006 Sep 19;45(37):11071-86. PMID:16964968
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