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J. Biol. Chem., Vol. 280, Issue 13, 12611-12620, April 1, 2005

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-Methylacyl-CoA Racemase from Mycobacterium tuberculosis

MUTATIONAL AND STRUCTURAL CHARACTERIZATION OF THE ACTIVE SITE AND THE FOLD^*

Kalle Savolainen, Prasenjit Bhaumik, Werner Schmitz¶, Tiina J. Kotti, Ernst Conzelmann¶, Rik K. Wierenga, and J. Kalervo Hiltunen||

From the Biocenter Oulu and Department of Biochemistry, University of Oulu, Linnanmaa, P. O. Box 3000, FIN-90014 University of Oulu, Finland and the ^¶Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany

-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of -methyl-branched CoA esters. Sequence comparisons have shown that this enzyme is a member of the family III CoA transferases. The mammalian Amacr is involved in bile acid synthesis and branched-chain fatty acid degradation. In human, mutated variants of Amacr have been shown to be associated with disease states. Amino acid sequence alignment of Amacrs and its homologues from various species revealed 26 conserved protic residues, assumed to be potential candidates as catalytic residues. Amacr from Mycobacterium tuberculosis (MCR) was taken as a representative of the racemases. To determine their importance for efficient catalysis, each of these 26 protic residues of MCR was mutated into an alanine, respectively, and the mutated variants were overexpressed in Escherichia coli. It was found that four variants (R91A, H126A, D156A, and E241A) were properly folded but had much decreased catalytic efficiency. Apparently, Arg⁹¹, His¹²⁶, Asp¹⁵⁶, and Glu²⁴¹ are important catalytic residues of MCR. The importance of these residues for catalysis can be rationalized by the 1.8 Å resolution crystal structure of MCR, which shows that the catalytic site is at the interface between the large and small domain of two different subunits of the dimeric enzyme. This crystal structure is the first structure of a complete enzyme of the bile acid synthesis pathway. It shows that MCR has unique structural features, not seen in the structures of the sequence related formyl-CoA transferases, suggesting that the family III CoA transferases can be subdivided in at least two classes, being racemases and CoA transferases.

Received for publication, August 24, 2004 , and in revised form, December 7, 2004.

^* This work was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, the Finnish Cultural Foundation, the Science Foundation of Instrumentarium and Research, and the Science Foundation of Farmos. 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 on-line version of this article (available at http://www.jbc.org) contains supplemental Table I and Figs. 1 and 2.

This article was selected as a Paper of the Week.

The atomic coordinates and structure factors (code 1X74) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

These authors contributed equally to this paper.

^|| To whom correspondence should be addressed. Tel.: 358-8-5531150; Fax: 358-8-5531141; E-mail: kalervo.hiltunen{at}oulu.fi.

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