OleTJE (CYP152L1) is a P450 peroxygenase that was first isolated from Jeotgalicoccus sp. 8456 in 2011. OleTJE is primarily a fatty acid decarboxylase, converting mid-chain fatty acids (C10:0 to C22:0) to terminal alkenes, which are industrially useful petrochemicals. Terminal alkenes are hydrophobic with high energy density, and are compatible with existing transportation infrastructure. Thus OleTJE has attracted considerable interest due to potential applications for generating "drop-in" biofuels. As a P450 peroxygenase, OleTJE is able to utilise H2O2 as a sole oxygen and hydrogen donor. This is atypical of P450s, which usually require electron transfer from redox partners to perform substrate oxidation. Other P450 peroxygenases have previously been characterised, including fatty acid hydroxylases P450 Spalpha (CYP152B1) from Sphingomonas paucimobilis and P450 BSβ (CYP152A1) from Bacillus subtilis. In addition to decarboxylation, OleTJE also hydroxylates fatty acids, generating 2-OH and 3-OH fatty acids as minor products. P450 BSβ has also been reported to perform low levels of decarboxylation. However, OleTJE has superior decarboxylase activity, posing questions about the mechanism of OleTJE. This thesis describes initial structural and biochemical characterisation of OleTJE. These data highlighted three amino acid residues thought to be key for effective catalysis: His85, Phe79 and Arg245. We hypothesised that the active site His85 could act as a proton donor to thereactive ferryl-oxo species compound I, allowing homolytic scission of the substrate C-Calpha bond to form the alkene product. Phe79 sandwiches His85 between the heme, and Arg245 co-ordinates the fatty acid carboxylate moiety. I performed mutagenesis studies to probe the roles of these residues, creating H85Q, F79A, F79W, F79Y, R245L and R245E OleTJE mutants, and characterised them by a combination of spectroscopic, analytical and structural methods. I also developed a novel system, where OleTJE was fused to alditol oxidase (AldO) from Streptomyces coelicolor, creating a fusion protein where addition of glycerol drives hydrogen peroxide production and the decarboxylation of fatty acids. Finally, studies showed that OleTJE is capable of performing secondary oxidation of hydroxylated products, which has expanded our knowledge of OleTJE's catalytic repertoire. This thesis also describes the initial characterisation of the OleTJE orthologue P450 KR from Kocuria rhizophila, which is also a terminal alkene-forming fatty acid decarboxylase. The crystal structure of P450 KR revealed an unusual dimeric state, with structural interactions unprecedented for a P450 enzyme. These data thus provide characterisation of two P450 peroxygenases involved in the production of terminal alkenes and which are of great interest as tools for the development of alternative sources of advanced biofuels.

Ellipticine is an alkaloid isolated from Apocynaceae plants exhibiting significant antitumor and anti-HIV activities. Cytochromes P450 (CYP) and peroxidases are the enzymes participating in metabolism of ellipticine. This process provides activation and detoxication metabolites of ellipticine. The CYP enzymes, which participate in oxidation of ellipticine in different tissues (liver, lung and kidney) of rat, a model organism simulating the fate of ellipticine in humans have already been identified. In this work, the effects of ellipticine on contents and catalytic activities of CYPs and other components of the mixed-function oxidase (MFO) system in this animal system were studied. For detection of contents of CYPs and other components of the MFO system, spectroscopic and electrochemical methods were used. To determine catalytic activities of CYPs and NADPH:cytochrome P450 reductase, reactions with specific substrates of these enzymes were utilized. The results found in this study demonstrate that expression and catalytic activity of CYP1A is induced by ellipticine in all of the tested organs (liver, kidney and lung) of rats treated with the drug. Moreover in liver, the cytochrome b5 expression is also induced. In addition, in this organ, expression and catalytic activity of CYP3A was increased by...

Richardson ground squirrels (gophers) are pests on the prairies that cause considerable agricultural and ecological damage. Traditional control methods such as the rodenticides strychnine, zinc phosphide, and anticoagulants, have proven ineffective in reducing gopher densities. In additional, current gopher control methods have the significant potential to cause primary and secondary toxicity to non-target animals. Thus, alternative methods for toxicological control of gophers are needed to mitigate these concerns. Present studies focused on the cytochrome P450 (CYP450) enzyme system responsible for xenobiotic detoxification in gophers. In vitro hepatic microsomal systems and HPLC analysis were used to elucidate general metabolic characteristics of major gopher xenobiotic metabolizing pathways. We found that the content and activity of individual components of the CYP450 system including CYP450, cytochrome b5, and NADPH-cytochrome P450 reductase in liver microsomal preparations were higher in gophers exposed to toxins used to control their population than in naïve (unexposed) gophers. When in vitro CYP450 mediated activities for five substrates [coumarin and aniline aromatic hydroxylation, 7-methoxycouamrin O-demethylation, and N-methylaniline, and N,N-dimethylaniline N-demethylation] were measured, naïve gophers were identified to have higher specific activity but similar whole body activity compared to the exposed gophers. Furthermore, there was a clearly identifiable sub-population of poor metabolizers showing considerably lower CYP450 activity within the gopher samples studied. Clotrimazole was found to be a potent inhibitor of several substrates of CYP450 enzyme-mediated reactions, which included aniline aromatic hydroxylation, N-methylaniline and N,N-dimethylaniline N-demethylation, and 7-methoxycoumarin O-demethylation. The cytotoxicity of above compounds was tested using freshly isolated gopher hepatocytes. The results showed that each compound caused considerable cytotoxicity to gopher hepatocytes. Addition of clotrimazole to the freshly isolated hepatocyte suspension increased the cytotoxicity of all tested compounds. <p>In conclusion, gophers may develop resistance to current chemical control methods through the enhancement of CYP450 system content, which can compensate the loss of enzyme activity. Furthermore, clotrimazole is a potent cytochrome P450 inhibitor, which increases the cytotoxicity caused by given compounds in gopher livers. The concept of using CYP450 enzyme inhibitor in combination with another chemical whose elimination depends on CYP450 metabolism to improve current gopher control method has practical importance.

Richardson ground squirrels (gophers) are pests on the prairies that cause considerable agricultural and ecological damage. Traditional control methods such as the rodenticides strychnine, zinc phosphide, and anticoagulants, have proven ineffective in reducing gopher densities. In additional, current gopher control methods have the significant potential to cause primary and secondary toxicity to non-target animals. Thus, alternative methods for toxicological control of gophers are needed to mitigate these concerns. Present studies focused on the cytochrome P450 (CYP450) enzyme system responsible for xenobiotic detoxification in gophers. In vitro hepatic microsomal systems and HPLC analysis were used to elucidate general metabolic characteristics of major gopher xenobiotic metabolizing pathways. We found that the content and activity of individual components of the CYP450 system including CYP450, cytochrome b5, and NADPH-cytochrome P450 reductase in liver microsomal preparations were higher in gophers exposed to toxins used to control their population than in naïve (unexposed) gophers. When in vitro CYP450 mediated activities for five substrates [coumarin and aniline aromatic hydroxylation, 7-methoxycouamrin O-demethylation, and N-methylaniline, and N,N-dimethylaniline N-demethylation] were measured, naïve gophers were identified to have higher specific activity but similar whole body activity compared to the exposed gophers. Furthermore, there was a clearly identifiable sub-population of poor metabolizers showing considerably lower CYP450 activity within the gopher samples studied. Clotrimazole was found to be a potent inhibitor of several substrates of CYP450 enzyme-mediated reactions, which included aniline aromatic hydroxylation, N-methylaniline and N,N-dimethylaniline N-demethylation, and 7-methoxycoumarin O-demethylation. The cytotoxicity of above compounds was tested using freshly isolated gopher hepatocytes. The results showed that each compound caused considerable cytotoxicity to gopher hepatocytes. Addition of clotrimazole to the freshly isolated hepatocyte suspension increased the cytotoxicity of all tested compounds. <p>In conclusion, gophers may develop resistance to current chemical control methods through the enhancement of CYP450 system content, which can compensate the loss of enzyme activity. Furthermore, clotrimazole is a potent cytochrome P450 inhibitor, which increases the cytotoxicity caused by given compounds in gopher livers. The concept of using CYP450 enzyme inhibitor in combination with another chemical whose elimination depends on CYP450 metabolism to improve current gopher control method has practical importance.

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High catalytic activity and a broad substrate range are characteristic of P450 fusion enzymes of the CYP102A class. P450 BM3 (CYP102A1, BM3) is a paradigm for the P450 fusion enzymes and is accredited with the highest monooxygenase activity in the P450 superfamily, a property which has led to its engineering and exploitation for biotechnologically valuable oxidation reactions. Initial research in the thesis focused on characterisation of a novel P450-redox partner fusion enzyme from the thermophilic fungus Myceliophthora thermophila (CYP505A30, P450MT1). Sequence alignments revealed a P450 domain and a diflavin P450 reductase domain with high sequence similarity to BM3’s domains (41% and 31% amino acid identity, respectively). The purified 118 kDa protein is soluble and exhibits characteristic P450 spectral properties, giving a Soret absorption shift to 450 nm upon binding CO to its ferrous heme iron. Binding titrations of intact P450 MT1 and its expressed P450 (heme) domain with fatty acid substrates and imidazole-based inhibitors revealed type I (blue) and II (red) Soret shifts, respectively, typical of other members of the P450 superfamily, and enabled determination of substrate binding constants. HPLC analysis confirmed stoichiometric amounts of bound FAD and FMN cofactors. Subsequent kinetic and biochemical studies included stopped-flow kinetic experiments showing that NADPH-dependent reduction of P450 MT1’s FAD cofactor occurs with a rate constant of ~150 s-1 at 20 °C. P450 MT1 has an unconventional substrate hydroxylation profile for saturated fatty acids. It hydroxylates these substrates predominantly at positions ω-1, ω-2 and ω-3. However, an unusual property of this enzyme is observed in its strong preference (~85% of total converted product) for either the ω-1 or the ω-2 position on odd and even chain length fatty acids, respectively. However, it displays higher selectivity for branched chain fatty acids over straight chain fatty acids, e.g. for the substrate iso-myristic acid, similar to BM3’s properties. Other work done focused on biophysical characterisation of the model P450-reductase fusion enzyme P450 BM3 from Bacillus megaterium. A combination of alternative structural techniques to X-ray crystallography were used to characterise the enzyme. More specifically, electron microscopy (EM) and nuclear magnetic resonance (NMR) were used to gain greater insights into the intimate associations of the enzyme monomers in BM3’s dimeric structure. These studies led to the first structural insights into how P450 BM3’s dimeric complex is organised. Dimerisation in BM3 arises predominantly from self-association of the enzyme’s FAD domains, and wild-type and mutant BM3 FAD domain forms were also characterised. Key FAD domain mutations that prevented intra-/inter-monomer disulphide bond formation facilitated the crystallization and determination of the FAD domain structure, the final part of the BM3 enzyme to have its three dimensional structure resolved. Data reported in this thesis give new insights into the biochemistry of biotechnologically important P450 monooxygenase enzymes from mesophilic and thermophilic microorganisms.

NADPH: cytochrome P450 oxidoreductase (POR) is an enzyme that is able to catalyze transfer of electrons from NADPH, via two-flavin cofactors, to various redox partners. Therefore, POR is essential for multiple metabolic processes, including reactions catalyzed by cytochromes P450. Due to all microsomal P450s depending on POR for the supply of electrons, disruption of POR may affect all microsomal P450 enzyme activities. Polymorphisms in human POR have been shown to lead to development phenotypes, the severity of which differs significantly depending on the degree of POR impairment. This thesis is focused on the preparation of POR, which is similar to combinatorial allele carrying two single nucleotide polymorphisms P228L and A503V, functionally not clearly characterized at that time. However, disastrous consequences have currently not been noted. Moreover, the presence of A503V has been confirmed as the most common allele, but there is evidence that A503V influences the activity of some redox partners. In present thesis there were two genes subcloned into expression plasmids pCW. The first of which carries the cDNA encoding the POR and the other carrying cDNA encoding POR with the histidine-tag. Expression of the recombinant POR was carried out in the heterologous bacterial system, using...

Plants are abundant in cytochrome P450s constituting about 1% of their protein coding genes. Some of these P450s catalyze oxidation reactions in metabolic pathways that lead to valuable compounds, like the anticancer drug paclitaxel, the blue pigment indigo and the promising antileukemic agent indirubin. The promiscuous nature of P450 catalysis enables simultaneous production of indirubin and indigo from a common substrate, but it also decreases the yield of paclitaxel in both plants and heterologous hosts, respectively. In this thesis, the catalytic promiscuity of CYP725A4 from Taxus cuspidata and CYP71B102 from Isatis tinctoria were investigated. CYP725A4 and CYP71B102 are involved in the biosynthesis of paclitaxel and indigo/indirubin pathways, respectively. CYP725A4 is known to catalyze the hydroxylation of endotaxadiene to taxadiene-5α-ol (T5α-ol), a precursor to paclitaxel, while CYP71B102 catalyzes the production of indigo and indirubin via hydroxylation of indole. CYP725A4 exhibited catalytic promiscuity upon heterologous expression in Escherchia coli producing 5(12)-oxa-3(11)-cyclotaxane (OCT) and 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as major products, and T5α-ol as a minor product with trace amounts of unidentified monooxygenated taxanes. The presence of T5α-ol was confirmed by comparing its gas chromatography and mass spectroscopy (GC-MS) retention time and spectrum with a standard T5α-ol, while those of others were verified by matching mass spectra from previous studies. Coexpression of CYP725A4 with cytochrome P450 reductase, CPR (as either fused or separate proteins) and cytochrome b5 (Cb5) did not affect the ratios of OCT, iso-OCT, and T5α-ol, although Cb5 had an apparently negative impact on CYP725A4 activity. Attempts to modify the catalytic promiscuity of CYP725A4 were conducted by mutating key residues at the active site of the enzyme. A mutant V3741 increased the production of T5α-ol by ~10 fold, although it still supported the formation of OCT and iso-OCT as major products. The levels of these compounds in V374I were almost 45% less than in native CYP725A4. Site-directed mutagenesis was also performed on taxadiene synthase (TS) to find a mutant that only produced exotaxadiene, which could be provided to CYP725A4 as an alternative substrate. Among the TS mutants generated, none were capable of producing only exotaxadiene, but two of the TS mutants, Y684C and Q609E, produced a reasonable amount of exotaxadiene. However, coexpression of these mutants with CYP725A4 and TCPR continued to produce OCT and iso-OCT. When CYP71B102 was expressed in E. coli, indigo was the main product, while indirubin and 2-oxindole were minor products, as verified by high-performance liquid chromatography (HPLC). Half-strength terrific broth (TB) medium in combination with 5-aminolevulinic acid supplementation and isatin hydrolase coexpression altogether increased indigo formation, while supplementation with isatin and 2-oxindole increased indirubin formation. The results of this study showed that the catalytic promiscuity of CYP725A4 and CYP71B102 could be modulated by metabolic and enzyme engineering to increase the yield of commercially important compounds, like paclitaxel, indigo and indirubin.

Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds remains problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. I have constructed several mutagenicity tester strains that co-express combinations of full-length human cytochrome P450 2E1, rat cytochrome P450 reductase, and human cytochrome b5 in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt-; and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. Coexpressing human cytochrome b5 with cytochrome P450 2E1 and cytochrome P450 reductase potentiates the mutagenicity observed with dialkylnitrosamines. These strains were sensitive to nitrosamines with varying alkyl side chains, including dimethylnitrosamine, diethylnitrosamine, dipropylnitrosamine, and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples, and may serve as useful tools in investigating the molecular properties of proteins in the cytochrome P450 monooxygenase system.