Aristolochene synthase: Probing the role of active site amino acids by site-directed mutagenesis

Aristolochene synthase is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl pyrophosphate (FPP) to (+)-aristolochene, the parent hydrocarbon of several fungal toxins. Two distinct aristolochene synthases have been isolated from the fungi Aspergillus terreus and Penicillium roqueforti. Both enzymes require Mg2+ as a cofactor. Extensive enzymological studies support a cyclization mechanism that proceeds through the formation of the cis-fused decalin (S)-(-)-germacrene A, although the proposed intermediate had never been detected during the formation of (+)-aristolochene.; The intermediacy of (S)-(-)-germacrene A was established by GC-MS examination of the pentane extracts derived from the incubation of FPP with wild-type P. roqueforti aristolochene synthase. This analysis revealed that in addition to (+)-aristolochene, a significant amount of (S)-(-)-germacrene A is released from the active site together with the side product (-)-valencene.; The highly conserved amino acids of the Mg2+ binding regions of the P. roqueforti enzyme, D115DVLE and N244DIYSYDKE, were altered by site-directed mutagenesis. The importance of these residues was established by an observed increase in Km and reduction in kcat of the mutants compared to the wild type. The function of these residues in guaranteeing the structural integrity of the active site necessary for the formation of the natural cyclization product (+)-aristolochene, was established by the analysis of the products generated by the mutant cyclases. The proportion of the intermediate (S)-(-)-germacrene A released from the mutants increased at the expense of (+)-aristolochene. The AS-E252Q mutant exclusively produced the intermediate (S)-(-)-germacrene A.; A GC-MS examination of the pentane extracts derived from the incubation of FPP with the A. terreus wild-type enzyme revealed that the cyclase exclusively produces (+)-aristolochene. A set of mutants of the A. terreus aristolochene synthase was constructed, targeting the residues of the magnesium-binding triad N219DIYSYEKE. The changes in steady state kinetic parameters of the mutants were similar to those observed for the corresponding mutants of the P. roqueforti enzyme. In addition, the mutants generated a large proportion of the intermediate (S)-(-)-germacrene at the expense of (+)-aristolochene.