Cytochrome P450-catalysed oxidations: Biosynthetic and mechanistic studies

The cytochromes P450 (P450s) comprise a superfamily of oxidative haemoproteins that catalyse a wide variety of oxidative transformations, during the course of which one atom from molecular oxygen is usually inserted into the substrate molecule. These transformations include but are not limited to hydrocarbon hydroxylation, heteroatom oxidation, alkene epoxidation, and oxidative carbon-carbon bond cleavage. Ubiquitous in nature, P450s are found in a diverse range of animals, bacteria, fungi, and plants. They are of particular interest for their involvement in biosynthetic and biodegradative processes (including steroid biosynthesis in animals, plants and fungi, and especially xenobiotic metabolism in animals), and significant attention has been focussed on investigating the mechanism(s) by which P450s mediate such a variety of transformations. The overall objective of this work was to gain a greater understanding of P450-mediated oxidations at a molecular level, using both in vivo and in vitro studies that are aimed at investigating particular aspects of certain reactions or enzyme-substrate systems. P450s are known to play crucial roles within several biosynthetic pathways in insects, as well as in pesticide degradation and resistance. Chapter 2 describes investigations into the major spiroacetal pheromone biosynthetic pathway of two Bactrocera fruit fly species, B. cacuminata and the highly pestiferous olive fly, B. oleae. This pathway has so far been shown to be identical within these species, and involves a number of oxidation steps that are (presumably) catalysed by P450s. The less pestiferous and locally available B. cacuminata provides a model species for studying this biosynthetic pathway in B. oleae, which is not found in Australia but causes a great deal of damage to olive plantations worldwide, particularly in the Mediterranean and North American regions. Disruption of B. oleae sex pheromone biosynthesis has been suggested as a possible species-specific pest control method, thus requiring a greater understanding of the pheromone biosynthetic pathway itself. A series of deuterium-labelled fatty acid-like compounds that could potentially serve as biosynthetic precursors to the major spiroacetal pheromone in vivo was conducted, followed by feeding experiments with B. cacuminata. This work has allowed delineation of the earlier steps in the biosynthetic pathway, and has established the inclusion of an enzyme-mediated carbon-carbon bond cleavage of a trioxygenated fatty acid equivalent as a key step. This process is likely to involve a P450 and appears to occur diastereoselectively via a threo vicinal diol intermediate. This is the first time such an oxidative transformation has been reported in insects, and as well as providing information specifically about pheromone biosynthesis in B. cacuminata (and presumably B. oleae), these investigations have also contributed to knowledge of the different ways in which spiroacetals are biosynthesised in insects. The mechanism(s) by which P450-mediated oxidations are carried out, and the identity of the active oxidant(s) in each case has been the subject of much, and often intense, debate. There is general consensus that the haem iron binds and activates molecular oxygen to form a ferryl species, known as Compound I, which is the major oxidising species. However, other species formed during the catalytic cycle are known to be involved in the mediation of certain transformations (e.g. oxidative C-C bond cleavage). Chapter 3 describes the synthesis of a number of structurally related fatty acids (including isotopically labelled compounds) designed to probe the mechanism(s) by which P450-mediated hydroxylation, sulfur oxidation, and epoxidation occur. The results of preliminary investigations into the oxidation of some of these compounds by the bacterial enzyme P450BM3 in vitro are also discussed. Steroids are also known to be substrates for a number of eukaryotic and prokaryotic P450s, both within biosynthetic and biodegradative pathways. Chapter 4 describes work aimed at investigating the structural features of the substrate that are essential for the regiospecific oxidation mediated by three steroid-metabolising P450s from Mycobacterium tuberculosis. This bacterium is the causative agent of tubercular infection in humans that results in millions of deaths annually. In addition, approximately one-third of the world’s population carries M. tuberculosis in a latent, non-infective state, and the emergence of drug-resistant strains has rendered the identification of new drug targets and the development of new treatments extremely important. Biodegradation of steroids by the enzymes CYP124A1, CYP125A1, and CYP142A1 provides carbon and energy for M. tuberculosis, and inhibition of these enzymes may provide a means of limiting bacterial growth, thus providing the development of alternate drug therapies. The synthesis of a number of side-chain analogues of cholesterol for use as mechanistic probes for these enzymes is described, and the results of their in vitro enzyme-mediated oxidation are discussed. This information has provided a greater understanding of the substrate structural features that are required for oxidation by each enzyme, as well as a starting point for the development of inhibitors specific to these bacterial P450s.