title: Pentalenolactone biosynthesis. Molecular cloning and assignment of biochemical function to PtlH, a non-heme iron dioxygenase of Streptomyces avermitilis.
abstract: The hydroxylase encoded by the ptlH (SAV2991) gene from the pentalenolactone gene cluster of Streptomyces avermitilis was cloned by PCR and expressed in Escherichia coli as an N-terminal His6-tag protein. Incubation of recombinant PtlH with (+/-)-1-deoxypentalenic acid (5) in the presence of Fe(II), alpha-ketoglutarate, and O2 gave (-)-11beta-hydroxy-1-deoxypentalenic acid (8), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat = 4.2 +/- 0.6 s-1 and Km (5) = 0.57 +/- 0.19 mM. 8 is a new intermediate in the conversion of the sesquiterpene pentalenene (3) to pentalenolactone (1).
authors.affiliation: Department of Chemistry, Brown University, Box H, Providence, Rhode Island 02912-9108, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: You
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; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D012717:Sesquiterpenes / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D012717:Sesquiterpenes; C017996:arenaemycin E; D049308:Dioxygenases