4luu | pdb_00004luu
V329A Epi-isozizaene synthase: Complex with Mg, inorganic pyrophosphate and benzyl triethyl ammonium cation
Structural highlights
Function[CYC1_STRCO] Catalyzes the cyclization of farnesyl diphosphate (FPP) to the sesquiterpene epi-isozizaene. Publication Abstract from PubMedThe class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-beta-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a beta-curcumene synthase, F95M EIZS is a beta-acoradiene synthase, F198L EIZS is a beta-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-gamma-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2-100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase.,Li R, Chou WK, Himmelberger JA, Litwin KM, Harris GG, Cane DE, Christianson DW Biochemistry. 2014 Feb 25;53(7):1155-68. doi: 10.1021/bi401643u. Epub 2014 Feb, 11. PMID:24517311[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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