Pentalenolactone Biosynthesis. Molecular Cloning and Assignment of Biochemical Function to PtlH, A Non-Heme Iron Dioxygenase of Streptomyces avermitilis

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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), α-ketoglutarate, and O2 gave (−)-11β-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).