3V1L

Crystal Structure of the S112A/H265Q mutant of a C-C hydrolase, BphD from Burkholderia xenovorans LB400

Summary for 3V1L

• Entry DOI: 10.2210/pdb3v1l/pdb
• Related: 2OG1 2PU7 2PUH 2PUJ 2RHT 2RHW 2RI6 3V1K 3V1M 3V1N
• Descriptor: 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase, MALONIC ACID (3 entities in total)
• Functional Keywords: c-c bond hydrolase, alpha/beta hydrolase fold, bphd, alpha/beta hydrolase, pcb degradation, meta cleavage product hydrolase, mcp hydrolase, 2-hydroxy-6-oxo-6-phenyl-hexa-2, 4-dienoate hydrolase, hydrolase
• Biological source: Burkholderia xenovorans
• Total number of polymer chains: 1
• Total formula weight: 32146.69

Authors

Ghosh, S.,Bolin, J.T. (deposition date: 2011-12-09, release date: 2012-03-21, Last modification date: 2024-02-28)

Primary citation

Ruzzini, A.C.,Ghosh, S.,Horsman, G.P.,Foster, L.J.,Bolin, J.T.,Eltis, L.D.
Identification of an Acyl-Enzyme Intermediate in a meta-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad.
J.Am.Chem.Soc., 134:4615-4624, 2012

PubMed Abstract: Meta-cleavage product (MCP) hydrolases are members of the α/β-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 Å resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of (18)O into the benzoate produced during hydrolysis in H(2)(18)O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES(red), previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.

PubMed: 22339283
DOI: 10.1021/ja208544g

Experimental method

X-RAY DIFFRACTION (2.11 Å)