title: Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis.
abstract: The PCR has been used to amplify a 2,181-bp ORF from Streptomyces coelicolor A3(2), designated SC9B1.20 (= SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase. The full-length recombinant protein was expressed at high levels in Escherichia coli and shown to catalyze the Mg(2+)-dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4S, 7R)-germacra-1 (10)E, 5E-diene-11-ol. The enzymatic cyclization had a k(cat) of 6.2 +/- 0.5 x 10(-3) s(-1) and a K(m) for farnesyl diphosphate of 62 +/- 8 nM. Expression of the N-terminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower k(cat) of 3.2 +/- 0.4 x 10(-3) s(-1) and a twofold greater k(m) of 115 +/- 14 nM. By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity. The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of Streptomyces species.
authors.affiliation: Department of Chemistry, Brown University, Providence, RI 02912, USA. David_Cane@brown.edu
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
authors.forename: Rory M
authors.initials: RM
authors.lastname: Watt
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D008401:Gas Chromatography-Mass Spectrometry; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009284:Naphthols / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D009284:Naphthols; D008025:Ligases; C001278:geosmin