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

摘要

The hydroxylase encoded by theptlH (SAV2991) gene from the pentalenolactone gene cluster ofStreptomyces avermitilis was cloned by PCR and expressed inEscherichia colias 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 of¹H,¹³C, COSY, HMQC, HMBC, and NOESY NMR. The steady state kinetic parameters werekcat 4.2±0.6 s⁻¹ andKm (>5) 0.57±0.19 mM.>8 is a new intermediate in the conversion of the sesquiterpene pentalenene (>3) to pentalenolactone (>1).