farnesyl-pyrophosphate and trichodiene

The sesquiterpenoid deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in specialized subcellular structures called toxisomes. The first step in DON synthesis is catalyzed by the sesquiterpene synthase (STS), Tri5 (trichodiene synthase), resulting in the cyclization of farnesyl diphosphate (FPP) to produce the sesquiterpene trichodiene. Tri5 is one of eight putative STSs in the F. graminearum genome. To better understand the F. graminearum terpenome, the volatile and soluble fractions of fungal cultures were sampled. Stringent regulation of sesquiterpene accumulation was observed. When grown in trichothecene induction medium, the fungus produces trichothecenes as well as several volatile non-trichothecene related sesquiterpenes, whereas no volatile terpenes were detected when grown in non-inducing medium. Surprisingly, a Δtri5 deletion strain grown in inducing conditions not only ceased accumulation of trichothecenes, but also failed to produce the non-trichothecene related sesquiterpenes. To test whether Tri5 from F. graminearum may be a promiscuous STS directly producing all observed sesquiterpenes, Tri5 was cloned and expressed in E. coli and shown to produce primarily trichodiene in addition to minor, related cyclization products. Therefore, while Tri5 expression in F. graminearum is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 does not explain the sesquiterpene deficient phenotype observed in the Δtri5 strain. To test whether Tri5 protein, separate from its enzymatic activity, may be required for non-trichothecene synthesis, the Tri5 locus was replaced with an enzymatically inactive, but structurally unaffected tri5

Topics: Carbon-Carbon Lyases; Cyclohexenes; Cytoplasmic Vesicles; Endoplasmic Reticulum; Fusarium; Mycotoxins; Polyisoprenyl Phosphates; Sesquiterpenes

The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.

Topics: Binding Sites; Carbon-Carbon Lyases; Catalysis; Chromatography, High Pressure Liquid; Cyclohexenes; Deuterium; Enzyme Stability; Escherichia coli; Kinetics; Lyases; Magnesium; Polyisoprenyl Phosphates; Recombinant Proteins; Sesquiterpenes

Trichodiene synthase catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon trichodiene. The enzyme normally requires a divalent cation, Mg2+, which can be substituted by Mn2+. Trichodiene synthase from Fusarium sporotrichioides has a highly conserved aspartate rich region, aa 100-104 (DDSKD). Three mutants were constructed by site-directed mutagenesis in which each aspartate residue was individually replaced by glutamate. The mutants were each overexpressed and purified to homogeneity. The importance of Asp100 and Asp101 for catalysis was established by the observation of an increase in Km as well as a reduction in kcat in the corresponding Glu mutants. Replacement of the Asp104 residue with Glu had little effect on either Km or kcat. All three mutants produced anomalous sesquiterpene products in addition to trichodiene when incubated with farnesyl diphosphate. Interestingly, when Mg2+ was replaced by Mn2+ in the incubation buffer, the kcat/Km of both wild type trichodiene synthase and the D104E dropped significantly, while those of the other two mutants were not much affected. The proportion of anomalous products increased significantly when the D100E and D101E mutants were incubated in the presence of Mn2+. These observations all lend weight to the proposal that the aspartate residues mediate substrate binding by chelation of the divalent metal ion. Asp100 and Asp101 appear to play a relatively more important role than Asp104.

Topics: Aspartic Acid; Binding Sites; Carbon-Carbon Lyases; Catalysis; Conserved Sequence; Cyclohexenes; Escherichia coli; Fusarium; Kinetics; Lyases; Magnesium; Manganese; Mutagenesis, Site-Directed; Polyisoprenyl Phosphates; Recombinant Proteins; Sesquiterpenes