title: Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-germacrene A synthase.
abstract: Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases.
authors.affiliation: School of Chemistry and ‡Cardiff Catalysis Institute, School of Chemistry, Cardiff University , Main Building, Park Place, Cardiff CF10 3AT, United Kingdom.
authors.forename: Veronica
authors.initials: V
authors.lastname: Gonzalez
authors.forename: Sabrina
authors.initials: S
authors.lastname: Touchet
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Grundy
authors.forename: Juan A
authors.initials: JA
authors.lastname: Faraldos
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D019295:Computational Biology; D003500:Cyclization; D015394:Molecular Structure; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism*; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D045788:Sesquiterpenes, Germacrane / Q000737:chemistry / Q000378:metabolism*; D036624:Solidago / Q000201:enzymology
chemical_list: D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C577204:germacrene A synthase, Solidago canadensis; C470416:germacrene D synthase