Pentalenolactone biosynthesis. Molecular cloning and assignment of biochemical function to PtlH, a non-heme iron dioxygenase of Streptomyces avermitilis

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).