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Albaflavenone synthase (CYP17OA1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptornyces coelicolor A3(2). Interestingly, CYP17OA1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand- free CYP17OA1 (2.6 Å) and complex of endogenous substrate (epiisozizaene) with CYP170A1 (3.3 Å). The structure of the complex suggests that the proximal episozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 a-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an a-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP17OA1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein. [ABSTRACT FROM AUTHOR]

The non-heme iron dioxygenase PtIH from the soil organism Streptomyces avermitilis is a member of the iron(II)/α-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtIH in several complexes with the cofactors iron, α-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 Å. resolution. The overall structure of PtIH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and α-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtIH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal α-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate. [ABSTRACT FROM AUTHOR]