title: Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases.
abstract: Terpenes are ubiquitous natural chemicals with diverse biological functions spanning all three domains of life. In specialized metabolism, the active sites of terpene synthases (TPSs) evolve in shape and reactivity to direct the biosynthesis of a myriad of chemotypes for organismal fitness. As most terpene biosynthesis mechanistically involves highly reactive carbocationic intermediates, the protein surfaces catalyzing these cascade reactions possess reactive regions possibly prone to premature carbocation capture and potentially enzyme inactivation. Here, we show using proteomic and X-ray crystallographic analyses that cationic intermediates undergo capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis.
authors.affiliation: Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology & Proteomics, The Salk Institute for Biological Studies , La Jolla, California 92037, United States.
authors.forename: Roland D
authors.initials: RD
authors.lastname: Kersten
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute , La Jolla, California 92037, United States.|Vincent J. Coates Mass Spectrometry Center, The Salk Institute of Biological Studies , La Jolla, California 92037, United States.
authors.forename: Jolene K
authors.initials: JK
authors.lastname: Diedrich
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute , La Jolla, California 92037, United States.|Vincent J. Coates Mass Spectrometry Center, The Salk Institute of Biological Studies , La Jolla, California 92037, United States.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.affiliation: Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology & Proteomics, The Salk Institute for Biological Studies , La Jolla, California 92037, United States.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000478:Alkylation; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009154:Mutation; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D013729:Terpenes / Q000737:chemistry
chemical_list: D013729:Terpenes; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase