2RI6

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

Replaces:  2PU6

Summary for 2RI6

• Entry DOI: 10.2210/pdb2ri6/pdb
• Related: 1C4X 1J1I 1U2E 2D0D 2OG1 2PU7
• Descriptor: 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase, SODIUM ION, MALONATE ION, ... (4 entities in total)
• Functional Keywords: hydrolase, c-c bond hydrolase, aromatic hydrocarbons catabolism
• Biological source: Burkholderia xenovorans
• Total number of polymer chains: 1
• Total formula weight: 31976.35

Authors

Bhowmik, S.,Bolin, J.T. (deposition date: 2007-10-10, release date: 2007-11-06, Last modification date: 2024-02-21)

Primary citation

Horsman, G.P.,Bhowmik, S.,Seah, S.Y.,Kumar, P.,Bolin, J.T.,Eltis, L.D.
The tautomeric half-reaction of BphD, a C-C bond hydrolase. Kinetic and structural evidence supporting a key role for histidine 265 of the catalytic triad.
J.Biol.Chem., 282:19894-19904, 2007

PubMed Abstract: BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (lambda(max) is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (lambda(max) is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (lambda(max) is 506 nm) with a similar rate, 1/tau approximately 500 s(-1). The crystal structure of the S112A:HOPDA complex at 1.8-A resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7 A away.

PubMed: 17442675
DOI: 10.1074/jbc.M702237200

Experimental method

X-RAY DIFFRACTION (1.68 Å)