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Please use this identifier to cite or link to this item: http://repository.iitr.ac.in/handle/123456789/26958
Title: 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
Authors: Horsman G.P.
Bhowmik S.
Seah S.Y.K.
Kumar, Pravindra R.Manish
Bolin J.T.
Eltis L.D.
Published in: Journal of Biological Chemistry
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 (λmax is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (λmax 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 (λmax is 506 nm) with a similar rate, 1/τ ∼ 500 s-1. The crystal structure of the S112A:HOPDA complex at 1.8-Å 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 Å away. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
Citation: Journal of Biological Chemistry, 282(27): 19894-19904
URI: https://doi.org/10.1074/jbc.M702237200
http://repository.iitr.ac.in/handle/123456789/26958
Issue Date: 2007
Keywords: BphD-catalyzed hydrolysis
Enol-keto tautomerization
Tautomeric half-reaction
Catalyst activity
Chemical bonds
Hydrolysis
Molecular interactions
Reaction kinetics
Enzyme kinetics
2 hydroxy 6 oxo 6 phenylhexa 2,4 dienoic acid
alkadiene
histidine
hydrolase
protein BphD
serine dehydratase
unclassified drug
article
catalysis
chemical bond
chemical reaction
complex formation
crystal structure
enzyme structure
molecular dynamics
priority journal
protein function
structure analysis
tautomeric shift
wild type
Bacterial Proteins
Burkholderia
Catalysis
Catalytic Domain
Crystallography, X-Ray
Fatty Acids, Unsaturated
Histidine
Hydrolases
Hydrolysis
Kinetics
Models, Molecular
Protein Structure, Quaternary
Burkholderia xenovorans
ISSN: 219258
Author Scopus IDs: 9846508100
17343445700
7006302128
55064809000
57197844041
7003863696
Author Affiliations: Horsman, G.P., Departments of Biochemistry and Molecular Biology, and Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
Bhowmik, S., Purdue Cancer Center, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2054, United States
Seah, S.Y.K., Departments of Biochemistry and Molecular Biology, and Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
Kumar, P., Purdue Cancer Center, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2054, United States
Bolin, J.T., Purdue Cancer Center, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2054, United States
Eltis, L.D., Departments of Biochemistry and Molecular Biology, and Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada, Dept. of Microbiology and Immunology, University of British Columbia, 1365-2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada
Funding Details: 
Corresponding Author: Eltis, L.D.; Dept. of Microbiology and Immunology, , Vancouver, BC V6T 1Z3, Canada; email: leltis@interchange.ubc.ca
Appears in Collections:Journal Publications [BT]

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