2og1
From Proteopedia
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'''Crystal Structure of BphD, a C-C hydrolase from Burkholderia xenovorans LB400''' | '''Crystal Structure of BphD, a C-C hydrolase from Burkholderia xenovorans LB400''' | ||
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[[Category: Ke, J.]] | [[Category: Ke, J.]] | ||
[[Category: 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase]] | [[Category: 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase]] | ||
| - | [[Category: | + | [[Category: Alpha/beta hydrolase]] |
| - | [[Category: | + | [[Category: Bphd]] |
| - | [[Category: | + | [[Category: Mcp hydrolase]] |
| - | [[Category: | + | [[Category: Meta cleavage product hydrolase]] |
| - | [[Category: | + | [[Category: Pcb degradation]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 10:49:52 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 07:49, 4 May 2008
Crystal Structure of BphD, a C-C hydrolase from Burkholderia xenovorans LB400
Overview
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 Structure
2OG1 is a Single protein structure of sequence from Burkholderia xenovorans. Full crystallographic information is available from OCA.
Reference
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 Page seeded by OCA on Sun May 4 10:49:52 2008
