TRICHODIENE SYNTHASE - IDENTIFICATION OF ACTIVE-SITE RESIDUES BY SITE-DIRECTED MUTAGENESIS

Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-C-14]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in k(cat)/K-m of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in K-m, as well as a 200-fold reduction in k(cat). In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on k(cat), while increasing the K-m by a factor of ca. 7-8. By contrast, the Y305T mutant exhibited an approximately 120-fold reduction in k(cat) as well as an 80-fold increase in K-m. Moreover, the Y305T enzyme converted farnesyl diphosphate to an approximately equal mixture of trichodiene and an unidentified sesquiterpene hydrocarbon.