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J. Biol. Chem., Vol. 279, Issue 30, 31312-31317, July 23, 2004

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Structure of 6-Oxo Camphor Hydrolase H122A Mutant Bound to Its Natural Product, (2S,4S)--Campholinic Acid

MUTANT STRUCTURE SUGGESTS AN ATYPICAL MODE OF TRANSITION STATE BINDING FOR A CROTONASE HOMOLOG^*

Philip M. Leonard and Gideon Grogan

From the York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom

The crotonase homolog, 6-oxo camphor hydrolase (OCH), catalyzes the desymmetrization of bicyclic -diketones to optically active keto acids via an enzymatic retro-Claisen reaction, resulting in the cleavage of a carbon-carbon bond. We have previously reported the structure of OCH (Whittingham, J. L., Turkenburg, J. P., Verma, C. S., Walsh, M. A., and Grogan, G. (2003) J. Biol. Chem. 278, 1744–1750), which suggested the involvement of five residues, His-45, His-122, His-145, Asp-154, and Glu-244, in catalysis. Here we report mutation studies on OCH that reveal that H145A and D154N mutants of OCH have greatly reduced values of k_cat/K_m derived from a very large increase in K_m for the native substrate, 6-oxo camphor. In addition, H122A has a greatly reduced value of k_cat, and its K_m is five times that of the wild-type. The location of the active site is confirmed by the 1.9-Å structure of the H122A mutant of OCH complexed with the minor diastereoisomer of (2S,4S)--campholinic acid, the natural product of the enzyme. This shows the pendant acetate of the product hydrogen bonded to a His-145/Asp-154 dyad and the endocyclic carbonyl of the cyclopentane ring hydrogen bonded to Trp-40. The results are suggestive of a base-catalyzed mechanism of C-C bond cleavage and provide clues to the origin of prochiral selectivity by the enzyme and to the recruitment of the crotonase fold for alternate modes of transition state stabilization to those described for other crotonase superfamily members.

Received for publication, March 30, 2004 , and in revised form, April 23, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1SZO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Recipient of a postdoctoral research fellowship from the Biotechnology and Biological Sciences Research Council (BBSRC).

To whom correspondence should be addressed. Tel.: 44-1904-328256; Fax: 44-1904-328266; E-mail: gg12{at}york.ac.uk.

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