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J. Biol. Chem., Vol. 280, Issue 7, 5456-5467, February 18, 2005

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Catalytic Mechanism of Chlamydia trachomatis Flavin-dependent Thymidylate Synthase^*

Jonathon Griffin, Christine Roshick, Emma Iliffe-Lee, and Grant McClarty¶

From the National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba R3E 3R2 and the Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada

Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H₄folate are products of the reaction. Production of H₄folate rather than H₂folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA^– E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH₂H₄folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and site-directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.

Received for publication, November 3, 2004 , and in revised form, December 7, 2004.

^* This work was supported by Grant GR-13301 from the Canadian Institutes of Health Research. 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.

^¶ To whom correspondence should be addressed: National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba R3E 3R2, Canada. Tel.: 204-789-6097; Fax: 204-789-2097; E-mail: mcclart{at}cc.umanitoba.ca.

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