title: Genome mining in Streptomyces clavuligerus: expression and biochemical characterization of two new cryptic sesquiterpene synthases.
abstract: Two presumptive terpene synthases of unknown biochemical function encoded by the sscg_02150 and sscg_03688 genes of Streptomyces clavuligerus ATCC 27074 were individually expressed in Escherichia coli as N-terminal-His₆-tag proteins, using codon-optimized synthetic genes. Incubation of recombinant SSCG_02150 with farnesyl diphosphate (1, FPP) gave (-)-δ-cadinene (2) while recombinant SSCG_03688 converted FPP to (+)-T-muurolol (3). Individual incubations of (-)-δ-cadinene synthase with [1,1-²H₂]FPP (1a), (1S)-[1-²H]-FPP (1b), and (1R)-[1-²H]-FPP (1c) and NMR analysis of the resulting samples of deuterated (-)-δ-cadinene supported a cyclization mechanism involving the intermediacy of nerolidyl diphosphate (4) leading to a helminthogermacradienyl cation 5. Following a 1,3-hydride shift of the original H-1(si) of FPP, cyclization and deprotonation will give (-)-δ-cadinene. Similar incubations with recombinant SSCG_03688 supported an analogous mechanism for the formation of (+)-T-muurolol (3), also involving a 1,3-hydride shift of the original H-1(si) of FPP.
authors.affiliation: Department of Chemistry, Brown University, Box H, Providence, RI 02912-9108, USA.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Hu
authors.forename: Wayne K W
authors.initials: WK
authors.lastname: Chou
authors.forename: Russell
authors.initials: R
authors.lastname: Hopson
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D003500:Cyclization; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016680:Genome, Bacterial / Q000235:genetics*; D023281:Genomics; D007700:Kinetics; D000081223:Polycyclic Sesquiterpenes; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism
chemical_list: D000081223:Polycyclic Sesquiterpenes; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C499351:T-muurolol; D013729:Terpenes; C054278:gamma-cadinene; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase