Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site

Abstract

Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 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 ligandfree CYP170A1 (2.6 Å ) and complex of endogenous substrate (epiisozizaene) with CYP170A1 (3.3 Å ). The structure of the complex suggests that the proximal epi-isozizaene 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 ␣-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 ␣-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 CYP170A1 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.Cytochrome P450 monooxygenases (CYP or P450) 2 are members of the most structurally diverse and functionally versatile superfamily of heme-containing enzymes with more than 10,000 known genes distributed among all biological kingdoms. CYP proteins have extremely diverse primary sequences and are grouped into different families when they have less than 40% amino acid sequence identity. Only the trans-cysteine ligand coordinating with the heme iron atom is conserved among all P450s (1). CYP enzymes are involved both in endogenous biosynthetic functions and in the metabolism of xenobiotic chemicals (2) and catalyze the oxidation of a wide variety of substrates with both regiospecificity and stereospecificity. There a few examples, however, of CYP enzymes that exhibit some degree of non-stereo-specific hydroxylation, for example, the oxidation of norbornane by CYP2B1 (3).Alabaflavenone monooxygenase was first identified from the soil bacterium Streptomyces coelicolor A3 (2), which is the most studied member of the Streptomyces genus of bacteria and produces a chemically diverse array of different secondary metabolites, including antibiotics, pigments, siderophores, hopanoids, and other lipids (4). CYP170A1 is a member of a two-gene operon also containing a sesquiterpene cyclase that converts farnesyl diphosphate (FPP) to the tricyclic hydrocarbon epi-isozizaene (5). This monooxygenase has been clearly shown to catalyze the conversion of...

Unusual Cytochrome P450 Enzymes and Reactions

“…At least three non-redox P450 reactions have been reported, including a phospholipase D-type hydrolysis by several mammalian P450s (51), pyrophosphatase (hydrolytic) activity of S. coelicolor CYP170A1 (52), and the rearrangement of a bicyclic pentaenone to an oxetane by CYP154A1 from the same bacterium (53). Those reactions are likely to involve elements of acid-base chemistry, but the details are unknown.…”

Unusual properties of the cytochrome P450 superfamily

“…This barrel is unusual because it consists of only four helices rather than the six found in all other terpene synthases. Mutagenesis established that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity [79]. The presence of two such distinct and unrelated biochemical activities in a single protein molecule is unprecedented within the P450 superfamily.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…61,62 This transformation itself does not seem to be physiologically relevant but perhaps may play a regulatory function as binding of farnesyl pyrophosphate, an albaavenone precursor, can alter the activity of the P450. 62 In contrast, CYP110C1, found in cyanobacterium Nostoc sp. strain PCC 7120 in an operon with a sesquiterpene cyclase, is reported to be crucial for formation of the carbon skeleton of the nal product (Fig.…”