In Taxus plants, the biosynthesis of the antimitotic pharmaceutical paclitaxel includes an acyl CoA-dependent acyltransferase that uses �-amino phenylpropanoyl CoAs to acylate a pathway precursor baccatin III. Several enzymes on the paclitaxel biosynthetic pathway are known, yet a few remain unidentified, including the putative ligase that biosynthesizes �-amino phenylpropanoyl CoAs. The mutlienzyme NRPS that produces tyrocidines contains a tridomain starter module tyrocidine synthetase A (TycA). TycA normally activates (S)-a-Phe to a (S)-a-Phe-AMP anhydride intermediate in the adenylation (A) domain. The Phe moiety is thenthioesterified by the pendent pantetheine of the adjacent thiolation (T) domain with release of AMP. Here, we showed that the A- and T-domains function as a CoA ligase, making a-, �-phenylalanyl-, and phenylisoserinyl CoA.The latter two are substrates of a phenylpropanoyltransferase on the paclitaxel biosynthetic pathway. Kinetic evaluation of the ligase reaction showed that the Phe-AMP intermediate is tightly bound to the A-domain. Further, evaluation of the reaction rates showed the thioesterification reaction was enzyme-catalyzed and not a fortuitous coupling of released Phe-AMP and CoA in solution, as suspected in earlier studies with similar multienzyme complexes.

Taxol biosynthesis: Tyrocidine synthetase A catalyzes the production of phenylisoserinyl CoA and other amino phenylpropanoyl thioesters

246th National Meeting (2013)

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