title: Pentalenolactone biosynthesis. Molecular cloning and assignment of biochemical function to PtlI, a cytochrome P450 of Streptomyces avermitilis.
abstract: A gene cluster encoding all of the enzymes for the biosynthesis of the antibiotic pentalenolactone (1) has recently been identified in Streptomyces avermitilis. The biosynthetic gene cluster contains the ptlI (SAV2999) gene which encodes a cytochrome P450 (CYP183A1). PtlI was cloned by PCR and expressed in Escherichia coli as a C-terminal His6-tag protein. Recombinant PtlI bound pentalenene (3) with high affinity (KD = 1.44 +/- 0.14 muM). Incubation of recombinant PtlI with (+/-)-3 in the presence of NADPH, E. coli flavodoxin and flavodoxin reductase, and O2 resulted in conversion to a single enantiomer of pentalen-13-al (7), by stepwise allylic oxidation via pentalen-13-ol (6). The steady-state kinetic parameters for the oxidation of pentalenene (3) to pentalen-13-ol (6) were kcat = 0.503 +/- 0.006 min-1 and Km = 3.33+/-0.62 muM for 3.
authors.affiliation: Department of Chemistry, Brown University, Box H, Providence, Rhode Island 02912-9108, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Quaderer
authors.forename: Satoshi
authors.initials: S
authors.lastname: Omura
authors.forename: Haruo
authors.initials: H
authors.lastname: Ikeda
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
mesh_terms: D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D005810:Multigene Family; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012717:Sesquiterpenes / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
chemical_list: D007527:Isoenzymes; D012717:Sesquiterpenes; C017996:arenaemycin E; D003577:Cytochrome P-450 Enzyme System