P450 Reactions Research Articles

Biomimetic Metabolism of Kaurenoic Acid Validated by Microsomal Reactions

The cytochrome P450 is a remarkable family of enzymes with widespread occurrence in animals and plants. In humans, they catalyze phase I metabolism reactions, such as oxidations and hydroxylations, playing a significant role in xenobiotics detoxification. Although mechanistic details of the catalytic cycle of cytochrome P450 are known, and human isoforms have their crystal structure solved, mimicking cytochrome P450 reactions using in vitro systems is still challenging. For this reason, biological assays using isolated enzymes or microsomal fractions are typically required to study the oxidative metabolism of new drugs before approval by regulatory agencies. Here we demonstrate that a series of metalloporphyrin catalysts generate the same oxidative product in vitro as a liver microsomal assay. The bioactive diterpene kaurenoic acid was chosen as a substrate model because it is a component of widely used phytomedicines in Brazil, such as guaco syrups and copaiba waxes. We evaluated the influence of reaction parameters such as catalyst type, oxygen donor type, solvent, and catalyst concentration on substrate consumption. Three products had their structures proposed by mass spectrometry, and among them, a dihydroxylated oxidation product was detected as a major metabolite after liver microsomal biotransformation and using porphyrin catalysts. We envision that the use of metalloporphyrins could be a useful strategy for biomimetic oxidation of kaurenic terpenes.

Expression and characterization of a novel cytochrome P450 enzyme from Variovorax paradoxus S110

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.

Redox reactions of heme proteins with flavonoids

Proteins containing heme groups perform a variety of important functions in living organisms. The heme groups are involved in catalyzing oxidation/reduction reactions, in electron transfer, and in binding small molecules, like oxygen or nitric oxide. Flavonoids, low molecular weight plant polyphenols, are ubiquitous components of human diet. They are also components of many plant extracts used in herbal medicine as well as of food supplements. Due to their relatively low reduction potential, flavonoids are prone to oxidation. This paper provides a review of redox reactions of various heme proteins, including catalase, some peroxidases, cytochrome P450, cytochrome c, myoglobin, and hemoglobin with flavonoids. Potential biological significance of these reactions is discussed, in particular when flavonoids are delivered to the body at pharmacological doses.

Synthetic Heme Selenolate: Spectral and Reactivity Differences from Synthetic Heme Thiolate

The relationship between the potent catalytic reactivity of cytochrome P450 and the electron-donating character of the axial thiolate ligand has long been investigated. Here, we synthesize the first synthetic iron porphyrin coordinated by selenolate (Se-SR), which is a better electron-donor than thiolate. At ordinary temperature, the spin state of heme selenolate Se-SR is lower (S = 3/2) than that of our previously synthesized SR complex (S = 5/2), which is structurally identical with complex Se-SR except that the axial ligand is thiolate. The N-O stretching mode of the NO- Se-SR complex clearly indicates that the electron-donating trans-effect of selenolate is larger than that of thiolate. A cyclic voltammogram of Se-SR revealed that the FeIII/FeIV redox couple of Se-SR is lower than that of SR, but the two potentials are unexpectedly identical in the case of the FeII/FeIII redox couple. The catalytic activity of complex Se-SR is 13-fold higher than that of SR complex in substituted phenol oxidation with cumene hydroperoxide, which may reflect the O-O bond cleavage rate. On the other hand, the results of alkane-alkene competitive oxidation indicate that the active intermediate derived from SR has a greater preference than that derived from Se-SR for oxidizing alkane, although Se-SR gave a higher total product yield. Thus, the active intermediate derived from heme thiolate is more effective than that derived from heme selenolate for alkane hydroxylation. This is consistent with the emergence in nature of heme thiolate structure, not heme selenolate, in cytochrome P450, based on its function in alkane hydroxylation.

Use of Specific Inhibitory Antibodies for P450 Reaction Phenotyping of Investigational Drugs

Identifying the CYP450 enzymes underlying metabolism of investigational drugs (i.e., P450 reaction phenotyping; PRP) is critical to the development of effective therapeutics. Information gained from such studies can: a) provide insight into structural modifications of test drugs in order to improve metabolic stability and; b) specify an estimate of drug‐drug interaction potential as well as that of interindividual and species differences in oxidative metabolism. CYP450 chemical inhibitors (e.g., a‐naphthoflavone and sulfaphenazole) are often used in reaction phenotyping studies but the outcome is sometimes equivocal due to the non‐specific effects of these chemical agents plus their capacity to undergo P450‐dependent catabolism. In contrast, inhibitory P450 antibodies offer high specificity and irreversibility, and may thus represent a superior approach for characterizing individual P450 enzyme involvement in drug metabolism. We recently developed a compilation of highly‐specific polyclonal and monoclonal antibodies to the CYP450s comprising the major oxidative drug‐metabolizing enzymes in human liver, including CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (aka CYP ImmunoInhibit antibodies). As shown here, each of these antibodies proved capable of eliciting > 80% inhibition of probe drug metabolism by the cognate P450 antigens in human liver microsomes (HLMx), while giving insignificant effects on different CYP450s (Figure 1). The inhibition observed with each of the six CYP450 antibodies was not only highly‐specific but was also dose‐dependent, with maximal inhibition (80–90%) achieved at IgG:microsomal protein ratios ranging from 0.1 mg/mg to 3 mg/mg (Figure 2). Control (pre‐immune) antibodies had no effect on substrate metabolism. Such results show that inhibitory antibodies coupled with HLMx represent an alternative and powerful approach to chemical inhibitors for evaluating involvement of specific P450 enzymes in the hepatic metabolism of investigational drugs. Interestingly, studies ongoing with CYP ImmunoInhibit antibodies and MetMax™ cryopreserved human hepatocytes, which are liver cells that have been permeabilized and cryopreserved, have given analogous results to those achieved with HLMx. The utilization of inhibitory antibodies with HLMx represents an accurate model for PRP of new therapeutics due to the specificity of these immunoreagents, and the irreversible nature of the inhibition achieved, an important benefit when working with metabolically‐stable test agents.Specific Inhibition of Microsomal Drug Metabolism by CYP ImmunoInhibit AntibodiesFigure 1Dose‐Response Inhibition of Microsomal Drug Metabolism by CYP ImmunoInhibit AntibodiesFigure 2

Redox Partners: Function Modulators of Bacterial P450 Enzymes

The superfamily of cytochrome P450 monooxygenases (P450s) is widespread in all kingdoms of life. Functionally versatile P450s are extensively involved in diverse anabolic and catabolic processes. P450s require electrons to be transferred by redox partners (RPs) for O2 activation and substrate monooxygenation. Unlike monotonic eukaryotic cytochrome P450 reductases, bacterial RP systems are more diverse and complicated. Recent studies have demonstrated that the type, the amount, the combination, and the mode of action of bacterial RPs can affect not only the catalytic rate and product distribution but also the type and selectivity of P450 reactions. These results are leading to a novel opinion that RPs not only function as auxiliary electron transfer proteins but are also important P450 function modulators.

Electrochemically boosted cytochrome P450 reaction that efficiently produces 25-hydroxyvitamin D3

We investigated electrochemical P450 vitamin D3 hydroxylase (Vdh) reactions using promiscuous spinach ferredoxin and a small organic molecule as the mediators that shuttle electrons from the electrode to the P450. The choice of small mediator and modified electrode surface were crucially important because they decrease electron loss to undesired reactions and reduce the amount of enzyme-deactivating reactive oxygen species produced. Twice the amount of 25-hydroxyvitamin D3 was produced under optimum conditions compared to that obtained by the conventional in vitro conversion system using NADH as the electron source, which demonstrates the electrochemical boost provided to the P450 reaction. We also confirmed that the present system works with crude extracts from Escherichia coli containing Vdh, which shows that it is possibly useful for a wide variety of P450 applications.

P27 - Association of cytochrome P450 reactivity and repeated-dose toxicity of chemical compounds

P27 - Association of cytochrome P450 reactivity and repeated-dose toxicity of chemical compounds

Application of Solanum lycopersicum Glucose-6-phosphate Dehydrogenase to NADPH-generating System for Cytochrome P450 Reactions

Cytochrome P450 (P450 or CYP) is involved in the metabolism of endogenous and exogenous compounds in most organisms. P450s have great potential as biocatalysts in the pharmaceutical and fine chemical industries because they catalyze diverse oxidative reactions using a wide range of substrates. The high-cost nicotinamide cofactor, NADPH, is essential for P450 reactions. Glucose-6-phosphate dehydrogenase (G6PDH) has been commonly used in NADPH-generating systems (NGSs) to provide NADPH for P450 reactions. Currently, only two G6PDHs from Leuconostoc mesenteroides and Saccharomyces cerevisiae can be obtained commercially. To supply high-cost G6PDH cost-effectively, we cloned the cytosolic G6PDH gene of Solanum lycopersicum (tomato) with 6xHis tag, expressed it in Escherichia coli, and purified the recombinant G6PDH (His-G6PDH) using affinity chromatography. In addition, enzymatic properties of His-G6PDH were investigated, and the His-G6PDH-coupled NGS was optimized for P450 reactions. His-G6PDH supported CYP102A1-catalyzed hydroxylation of omeprazole and testosterone by NADPH generation. This result suggests that tomato His-G6PDH could be a cost-effective enzyme source for NGSs for P450-catalyzed reactions as well as other NADPH-requiring reactions.

Application of cytochrome P450 reactivity on the characterization of chemical compounds and its association with repeated-dose toxicity

Repeated-dose toxicity (RDT) studies are one of the critical studies to assess chemical safety. There have been some studies attempting to predict RDT endpoints based on chemical substructures, but it remains very difficult to establish such a method, and a more detailed characterization of chemical compounds seems necessary. Cytochrome P450s (P450s) comprise multiple forms with different substrate specificities and play important roles in both the detoxification and metabolic activation of xenobiotics. In this study, we investigated possible use of P450 reactivity of chemical compounds to classify the compounds. A total of 148 compounds with available rat RDT test data were used as test compounds and subjected to inhibition assays against 18 human and rat P450s. Among the tested compounds, 82 compounds inhibited at least one P450 form. Hierarchical clustering analyses using the P450 inhibitory profiles divided the 82 compounds into nine groups, some of which showed characteristic chemical and biological properties. Principal component analyses of the P450 inhibition data in combination with the calculated chemical descriptors demonstrated that P450 inhibition data were plotted differently than most chemical descriptors in the loading plots. Finally, association analyses between P450 inhibition and RDT endpoints showed that some endpoints related to the liver, kidney and hematology were significantly associated with the inhibition of some P450s. Our present results suggest that the P450 reactivity profiles can be used as novel descriptors for characterizing chemical compounds for the investigation of the toxicity mechanism and/or the establishment of a toxicity prediction model.

Kinetic Modeling of Steady-State Situations in Cytochrome P450 Enzyme Reactions.

In the course of investigations of the kinetics of individual reactions of cytochrome P450 (P450) enzymes, a number of points about the complexity of P450 enzyme kinetics have become apparent. Several of these are of particular relevance to work with P450 enzymes in the course of drug development and lead optimization, particularly with regard to estimating in vitro kinetic parameters and dealing with enzyme inhibitors. Modern simulation modeling has been applied to situations involving issues of preincubation time with moderate strength and strong inhibitors, inhibition by tightly bound ligands that have been identified in P450 enzymes, extensive substrate depletion, P450 reactions with a rate-limiting step after product formation, and the consumption of an inhibitor during a reaction by either a P450 enzyme being monitored or another one in a mixture. The results all follow from first principles, and simulations reveal the extent of their significance in various settings. The order of addition of substrate and inhibitors can change the apparent outcome (inhibition constant, K i), and the effect of the order is more pronounced with a stronger inhibitor. Substrate depletion alters parameters (Michaelis constant, K m) and can generate apparently sigmoidal plots. A rate-limiting step after product formation lowers the apparent K m and distorts K i Consumption of an inhibitor during a reaction affects K i and differs depending on which enzyme is involved. The results are relevant with P450 enzymes and other enzymes as well. SIGNIFICANCE STATEMENT: Kinetic simulations have been used to address several potential problems in enzyme kinetic analysis. Although the simulations done here are general for enzyme reactions, several problems addressed here are particularly relevant to cytochrome P450 reactions encountered in drug development work.

Defining optimal electron transfer partners for light-driven cytochrome P450 reactions

Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.

An Extensively Humanized Mouse Model to Predict Pathways of Drug Disposition and Drug/Drug Interactions, and to Facilitate Design of Clinical Trials.

Species differences in drug metabolism and disposition can confound the extrapolation of in vivo PK data to man and also profoundly compromise drug efficacy studies owing to differences in pharmacokinetics, in metabolites produced (which are often pharmacologically active), and in differential activation of the transcription factors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), which regulate the expression of such enzymes as P450s and drug transporters. These differences have gained additional importance as a consequence of the use of genetically modified mouse models for drug-efficacy testing and also patient-derived xenografts to predict individual patient responses to anticancer drugs. A number of humanized mouse models for cytochrome P450s, CAR, and PXR have been reported. However, the utility of these models has been compromised by the redundancy in P450 reactions across gene families, whereby the remaining murine P450s can metabolize the compounds being tested. To remove this confounding factor and create a mouse model that more closely reflects human pathways of drug disposition, we substituted 33 murine P450s from the major gene families involved in drug disposition, together with Car and Pxr, for human CAR, PXR, CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP3A4, and CYP3A7. We also created a mouse line in which 34 P450s were deleted from the mouse genome. Using model compounds and anticancer drugs, we demonstrated how these mouse lines can be applied to predict drug-drug interactions in patients and discuss here their potential application in the more informed design of clinical trials and the personalized treatment of cancer.

Sequential testicular atrophy involves changes in cellular proliferation and apoptosis associated with variations in aromatase P450 expression levels in Irs-2-deficient mice.

Insulin receptor substrate 2 (Irs-2) is an intracellular protein susceptible to phosphorylation after activation of the insulin receptor. Its suppression affects testis development and its absence induces peripheral resistance to insulin. The aim of this study was to identify changes induced by the deletion of Irs-2 in the testicular structure and by the altered expression of cytochrome P450 aromatase, a protein necessary for the development and maturation of germ cells. Adult knockout (KO) mice (Irs-2-/- , 6 and 12weeks old) and age-matched wild-type (WT) mice were used in this study. Immunohistochemistry and Western blot analyses were performed to study proliferation (PCNA), apoptosis (active caspase-3) and P450 aromatase expression in testicular histological sections. Deletion of Irs-2 decreased the number of epithelial cells in the seminiferous tubule and rete testis. Aberrant cells were frequently detected in the epithelia of Irs-2-/- mice, accompanied by variations in spermatogonia, which were shown to exhibit small hyperchromatic nuclei as well as polynuclear and anuclear structures. The amount of cell proliferation was significantly lower in Irs-2-/- mice than in WT mice, whereas apoptotic processes were more common in Irs-2-/- mice. Aromatase P450 reactivity was higher in 6-week-old KO mice than in WT mice of the same age and was even higher at 12weeks. Our results suggest that Irs-2 is a key element in spermatogenesis because silencing Irs-2 induces the sequential development of testicular atrophy. The effects are observed mainly in germ cells present in the seminiferous tubule, which may be due to changes in cytochrome P450 aromatase expression.

Mechanistic Insights into Interactions between Bacterial Class I P450 Enzymes and Redox Partners

Cytochrome P450 enzymes are highly diversified biocatalysts associated with steroid biosynthesis, xenobiotic metabolism, biosynthesis of natural products, and industrial oxidation reactions. A typical P450 catalytic cycle requires sequential transfer of two electrons from NAD(P)H to the heme-iron reactive center for O2 activation. For the most abundant bacterial Class I P450 systems, this important process is usually mediated by two redox partner proteins including an FAD-containing ferredoxin reductase (FdR) and a small iron–sulfur protein, ferredoxin (Fdx). However, it is often unclear which pair of Fdx and FdR among multiple redox partners is the optimal one for a specific Class I P450 enzyme. To address this important but underexplored question, herein, a reaction matrix network with 16 Fdxs, 8 FdRs, and 6 P450s (against 7 substrates) was constituted. By analyzing the reactivity profiles of 896 P450 reactions, together with phylogenetic analysis, redox potential measurements, structural simulations, a...

A tailor-made, self-sufficient and recyclable monooxygenase catalyst based on coimmobilized cytochrome P450 BM3 and glucose dehydrogenase.

Cytochrome P450 monooxygenases (P450s) promote hydroxylations in a broad variety of substrates. Their prowess in C–H bond functionalization renders P450s promising catalysts for organic synthesis. However, operating P450 reactions involve complex management of the main substrates, O2 and nicotinamide adenine dinucleotide phosphate (NAD(P)H) reducing equivalents against an overall background of low operational stability. Whole‐cell biocatalysis, although often used, offers no general solution to these problems. Herein, we present the design of a tailor‐made, self‐sufficient, operationally stabilized and recyclable P450 catalyst on porous solid support. Using enzymes as fusion proteins with the polycationic binding module Zbasic2, the P450s BM3 (from Bacillus megaterium) was coimmobilized with glucose dehydrogenase (type IV; from B. megaterium) on anionic sulfopropyl‐activated carrier (ReliSorb SP). Immobilization via Zbasic2 enabled each enzyme to be loaded in controllable amount, thus maximizing the relative portion of the rate limiting P450 BM3 (up to 19.5 U/gcarrier) in total enzyme immobilized. Using lauric acid as a representative P450 substrate that is poorly accessible to whole‐cell catalysts, we demonstrate complete hydroxylation at low catalyst loading (≤0.1 mol%) and efficient electron coupling (74%), inside of the catalyst particle, to the regeneration of NADPH from glucose (27 cycles) was achieved. The immobilized P450 BM3 showed a total turnover number of ∼18,000, thus allowing active catalyst to be recycled up to 20 times. This study therefore supports the idea of practical heterogeneous catalysis by P450s systems immobilized on solid support.

Disposition and metabolism of the G protein-coupled receptor 40 agonist TAK-875 (fasiglifam) in rats, dogs, and humans

The absorption, distribution, metabolism, and excretion of fasiglifam were investigated in rats, dogs, and humans.The absolute oral bioavailability of fasiglifam was high in all species (>76.0%).After oral administration of [14C]fasiglifam, the administered radioactivity was quantitatively recovered and the major route of excretion of radioactivity was via feces in all species.Fasiglifam was a major component in the plasma and feces in all species. Its oxidative metabolite (M-I) was observed as a minor metabolite in rat and human plasma (<10% of plasma radioactivity). In human plasma, hydroxylated fasiglifam (T-1676427), the glucuronide of fasiglifam (fasiglifam-G), and the glucuronide of M-I were detected as additional minor metabolites (<2% of plasma radioactivity). None of these metabolites were specific to humans. Fasiglifam-G was the major component in the rat and dog bile.In vitro cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) reaction phenotyping indicated that oxidation (to form M-I and T-1676427) and glucuronidation of fasiglifam are mainly mediated by CYP3A4/5 and UGT1A3, respectively.Fasiglifam and fasiglifam-G are substrates of BCRP and Mrp2/MRP2, respectively.Glucuronidation of fasiglifam-G was found to be the predominant elimination pathway of fasiglifam in all species tested, including humans.

Molecular Mechanism of Alternative P450-Catalyzed Metabolism of Environmental Phenolic Endocrine-Disrupting Chemicals.

Understanding the bioactivation mechanisms to predict toxic metabolites is critical for risk assessment of phenolic endocrine-disrupting chemicals (EDCs). One mechanism involves ipso-substitution, which may contribute to the total turnover of phenolic EDCs, yet the detailed mechanism and its relationship with other mechanisms are unknown. We used density functional theory to investigate the P450-catalyzed ipso-substitution mechanism of the prominent xenoestrogen bisphenol A. The ipso-substitution proceeds via H-abstraction from bisphenol A by Compound I, followed by essentially barrierless OH-rebound onto the ipso-position forming a quinol, which can spontaneously decompose into the carbocation and hydroquinone. This carbocation can further evolve into the highly estrogenic hydroxylated and dimer-type metabolites. The H-abstraction/OH-rebound reaction mechanism has been verified as a general reaction mode for many other phenolic EDCs, such as bisphenol analogues, alkylphenols and chlorophenols. The identified mechanism enables us to effectively distinguish between type I (eliminating-substituent as anion) and type II (eliminating-substituent as cation) ipso-substitution in various phenolic EDCs. We envision that the identified pathways will be applicable for prediction of metabolites from phenolic EDCs whose fate are affected by this alternative type of P450 reactivity, and accordingly enable the screening of these metabolites for endocrine-disrupting activity.

Substrate Channeling of Prostaglandin H2 on the Stereochemical Control of a Cascade Cyclization Route

Enzymes can carry out effective rate accelerations by virtue of their ability to utilize substrate-channeling forces to act as a mechanochemical valve. Such a channeling process is treated quantitatively using the key aspects of the free energy landscape; the balance between substrate positioning and conformational changes reflects the severe geometric and electronic requirements for the relatively tight transition state. The observed kcat/KM of about 106 M–1 s–1 for PGH2 cyclization has been revealed to be brought about by bringing together two properly oriented reactants of substrate and enzyme regarding the magnitude significance of the contribution from outer- and inner-binding stereopopulation along the free-energy channeling pathway and thus shapes the cascade cyclization route, enforcing precise spatial and temporal control. The apparent constant kcat of many P450 reactions involving the heme catalytic cycle, which is often on the order of 101–102 s–1 and is usually attributed to compound 0 to comp...

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

Covering: 2000 up to 2018 The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C-H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations. In this review, we aim to provide an overview of the range of chemical transformations that P450 enzymes can catalyse within bacterial secondary metabolism, with the intention to provide an important resource to aid in understanding of the potential roles of P450 enzymes within newly identified bacterial biosynthetic pathways.