title: Improved selectivity of an engineered multi-product terpene synthase.
abstract: Mutation of the sesquiterpene synthase Cop2 was conducted with a high-throughput screen for the cyclization activity using a non-natural substrate. A mutant of Cop2 was identified that contained three amino acid substitutions. This mutant, 17H2, converted the natural substrate FPP into germacrene D-4-ol with 77% selectivity. This selectivity is in contrast to that of the parent enzyme in which germacrene D-4-ol is produced as 29% and α-cadinol is produced as 46% of the product mixture. The mutations were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a kcat/KM of 0.62 mM(-1) s(-1), which is nearly identical to that of the parent Cop2, which had a kcat/KM of 0.58 mM(-1) s(-1).
authors.affiliation: California Institute of Technology, Division of Chemistry & Chemical Engineering, 1200 E. California Blvd., Pasadena CA 91125, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Lauchli
authors.forename: Julia
authors.initials: J
authors.lastname: Pitzer
authors.forename: Rebekah Z
authors.initials: RZ
authors.lastname: Kitto
authors.forename: Karolina Z
authors.initials: KZ
authors.lastname: Kalbarczyk
authors.forename: Kersten S
authors.initials: KS
authors.lastname: Rabe
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D003302:Coprinus / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D016415:Sequence Alignment; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase