Pentalenolactone Biosynthesis. Molecular Cloning and Assignment of Biochemical Function to PtlI, a Cytochrome P450 of Streptomyces avermitilis

• Published in: Journal of the American Chemical Society 2006-10, Vol.128 (40), p.13036-13037
• Main Authors: Quaderer, Richard, Omura, Satoshi, Ikeda, Haruo, Cane, David E
• Format: Article
• Language: English
• Subjects: Cloning, Molecular; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Isoenzymes - genetics; Isoenzymes - metabolism; Kinetics; Multigene Family; Open Reading Frames; Polymerase Chain Reaction; Sesquiterpenes - metabolism; Streptomyces - enzymology; Streptomyces - genetics
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0639214

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 (K D = 1.44 ± 0.14 μM). 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 k cat = 0.503 ± 0.006 min-1 and K m = 3.33±0.62 μM for 3.