Catalytic mechanism of Chlamydia trachomatis flavin-dependent thymidylate synthase.

摘要: Here we report on a Chlamydia trachomatis gene that complements the growth defect of thymidylate synthase-deficient strain Escherichia coli. The complementing encodes 60.9-kDa protein shows low level primary sequence homology to new class thymidylate-synthesizing enzymes, termed flavin-dependent synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates CTThyX is active as tetramer NADPH, methylenetetrahydrofolate, and dUMP substrates, serving source reducing equivalents, methyl donor, acceptor, respectively. dTMP H(4)folate products reaction. Production rather than H(2)folate, in classical synthase reaction, eliminates need dihydrofolate reductase, explaining trimethoprim-resistant phenotype displayed by thyA(-) E. coli-expressing CTThyX. In contrast extensively characterized thyA-encoded synthases, which form ternary complex substrates CH(2)H(4)folate follow an ordered sequential mechanism, follows ping-pong kinetic mechanism involving enzyme intermediate. Mass spectrometry was used localize group arginine, mutagenesis showed this be critical synthesizing activity. These differentiating characteristics clearly distinguish from ThyA, making enzymes attractive targets rational drug design.