Structural and Mechanistic Analysis of Trichodiene Synthase using Site-Directed Mutagenesis: Probing the Catalytic Function of Tyrosine-295 and the Asparagine-225/Serine-229/Glutamate-233–Mg2+B Motif

摘要

Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the “aspartate-rich” motif >D¹⁰⁰DXX(D/E) that coordinates to Mg²⁺A and Mg²⁺C, and the “NSE/DTE” motif >N²²⁵DXX>SXXX>E that chelates Mg²⁺B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like >DDXX>TXXX>E motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.