Microbial P450s Research Articles

Recombinant expression and characterization of novel P450s from Actinosynnema mirum

Cytochrome P450 monooxygenases (P450s) are the major contributor in the metabolism of xenobiotics, including therapeutic agents. Thus, P450s find broad application in the pharmaceutical industry to synthesize metabolites of new active pharmaceutical ingredients in order to evaluate toxicity and pharmacokinetics. As an alternative to human hepatic P450s, microbial P450s offer several advantages, such as an easier and more efficient heterologous expression as well as higher stability under process conditions. Recently, the wild-type strain Actinosynnema mirum has been reported to catalyze hydroxylation reactions with high activity on a broad range of substrates. In this study, one of these substrates, ritonavir, was used to analyze the transcriptional response of the wild-type strain. Analysis of the differential gene expression pattern allowed the assignment of genes potentially responsible for ritonavir conversion. Heterologous expression of these candidates and activity testing led to the identification of a novel P450 that efficiently converts ritonavir resembling the activity of the human CYP3A4.

Bioactive Indanes: Comparative in vitro Metabolism Study of PH46A, a New Potential Anti-inflammatory Agent and Biosynthesis of its Primary Metabolite PH132

PH46A is the lead of a new class of bioactive indanes with potential for the treatment of inflammatory bowel disease. A qualitative in vitro metabolism profile of PH46A was investigated in preclinical studies, and the rate of its metabolism in cryopreserved hepatocytes prepared from male Sprague Dawley rat, Beagle dog, Cynomolgus monkey and pooled mixed gender human was compared by LC-MS. The clearance order of PH46A was determined to be rat>dog>monkey>human. The species tested which exhibited the closest clearance values to the human was monkey. Following incubation of PH46A with cryopreserved hepatocytes, 5 metabolites were identified, including M1 (keto-PH46), M2 (PH46-OH, PH132), M3 (PH46-diOH), M4 (keto-glucuronide-PH46) and M5 (glucuronide conjugate-PH46). It was found that the human metabolites M2 and M5 were also present in rat, dog and monkey, while M1 was present in all species except monkey. M2 was detected in dog and monkey by LC1 conditions, but only in dog by LC2. Therefore, the metabolism in rat was most similar to human, in terms of the metabolites observed, but all putative human metabolites were present in rat and dog. We further explored the characterization of key metabolite M2 (PH46-OH). Identical PH46-OH was obtained via a bio-catalytic oxidation method from PH46 using rat liver microsomes (RLM) and the human liver P450s (Cyp2D6, Cyp 2C19 and Cyp 4A11) following screening of selectAZyme panels of microbial P450s, recombinant human liver P450s and different microsomes. RLM was used in scale up production and PH132 was isolated and characterized as 4-(((1’S,2’S)-1’,6-dihydroxy-1’,3’-dihydro1H,2’H-[2,2’-biinden]-2’-yl)methyl) benzoic acid via LC-MS/MS, NMR and HRMS. The site of hydroxylation on the biindane scaffold was unexpected. The outcomes of these studies have provided valuable information for future pharmacokinetic and in vivo toxicological investigations.

Microbial P450 Enzymes in Bioremediation and Drug Discovery: Emerging Potentials and Challenges.

Cytochrome P450 enzymes are a structurally conserved but functionally diverse group of heme-containing mixed function oxidases found across both prokaryotic and eukaryotic forms of the microbial world. Microbial P450s are known to perform diverse functions ranging from the synthesis of cell wall components to xenobiotic/drug metabolism to biodegradation of environmental chemicals. Conventionally, many microbial systems have been reported to mimic mammalian P450-like activation of drugs and were proposed as the in-vitro models of mammalian drug metabolism. Recent reports suggest that native or engineered forms of specific microbial P450s from these and other microbial systems could be employed for desired specific biotransformation reactions toward natural and synthetic (drug) compounds underscoring their emerging potential in drug improvement and discovery. On the other hand, microorganisms particularly fungi and actinomycetes have been shown to possess catabolic P450s with unusual potential to degrade toxic environmental chemicals including persistent organic pollutants (POPs). Wood-rotting basidiomycete fungi in particular have revealed the presence of exceptionally large P450 repertoire (P450ome) in their genomes, majority of which are however orphan (with no known function). Our pre- and post-genomic studies have led to functional characterization of several fungal P450s inducible in response to exposure to several environmental toxicants and demonstration of their potential in bioremediation of these chemicals. This review is an attempt to summarize the postgenomic unveiling of this versatile enzyme superfamily in microbial systems and investigation of their potential to synthesize new drugs and degrade persistent pollutants, among other biotechnological applications.

Terpene hydroxylation with microbial cytochrome P450 monooxygenases.

Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes.

Conversions of tricyclic antidepressants and antipsychotics with selected P450s from Sorangium cellulosum So ce56.

Human cytochromes P450 (P450s) play a major role in the biotransformation of drugs. The generated metabolites are important for pharmaceutical, medical, and biotechnological applications and can be used for derivatization or toxicological studies. The availability of human drug metabolites is restricted and alternative ways of production are requested. For this, microbial P450s turned out to be a useful tool for the conversion of drugs and related derivatives. Here, we used 10 P450s from the myxobacterium Sorangium cellulosum So ce56, which have been cloned, expressed, and purified. The P450s were investigated concerning the conversion of the antidepressant drugs amitriptyline, clomipramine, imipramine, and promethazine; the antipsychotic drugs carbamazepine, chlorpromazine, and thioridazine, as well as their precursors, iminodibenzyl and phenothiazine. Amitriptyline, chlorpromazine, clomipramine, imipramine, and thioridazine are efficiently converted during the in vitro reaction and were chosen to upscale the production by an Escherichia coli-based whole-cell bioconversion system. Two different approaches, a whole-cell system using M9CA medium and a system using resting cells in buffer, were used for the production of sufficient amounts of metabolites for NMR analysis. Amitriptyline, clomipramine, and imipramine are converted to the corresponding 10-hydroxylated products, whereas the conversion of chlorpromazine and thioridazine leads to a sulfoxidation in position 5. It is shown for the first time that myxobacterial P450s are efficient to produce known human drug metabolites in a milligram scale, revealing their ability to synthesize pharmaceutically important compounds.

Cholesterol, an essential molecule: diverse roles involving cytochrome P450 enzymes

Cholesterol is an essential molecule for eukaryotic life and is an important precursor for a wide range of physiological processes. Biosynthesis and homoeostasis of cholesterol are complex mechanisms that are tightly regulated and interlinked with activities of a number of cytochrome P450 enzymes. These P450s play central critical roles in cholesterol metabolism. Key roles include a rate-limiting reaction in the synthesis of cholesterol itself, and in the oxidative transformations of cholesterol into steroid hormones and bile acids. However, microbial P450s also have important roles that impinge directly on human cholesterol synthesis and oxidation. Recent data reveal that Mycobacterium tuberculosis (which infects more than one-third of the world's human population) uses P450s to initiate breakdown of host cholesterol as an energy source. Microbial P450s also catalyse industrially important transformations in the synthesis of cholesterol-lowering statin drugs, with clear benefits to humans. The present article reviews the various roles of P450s in human cholesterol metabolism, from endogenous P450s through to microbial oxidases that enable catabolism of human cholesterol, or facilitate production of statins that regulate cholesterol production with positive outcomes in cardiovascular disease.

The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism

Cytochrome P450 monooxygenases (P450s) are a diverse collection of enzymes acting on various endogenous and xenobiotic molecules. Most of them catalyse hydroxylation reactions and one group of possible substrates are fatty acids and their related structures. In this minireview, the significance of P450s in microbial fatty acid conversion is described. Bacteria and yeasts possess various P450 systems involved in alkane and fatty acid degradation, and often several enzymes with different activities and specificities are retrieved in one organism. Furthermore, P450s take part in the formation of fatty acid-based secondary metabolites. Finally, there are a substantial number of microbial P450s displaying activity towards fatty acids, but to which no biological role could be assigned despite the often quite intense research.

Mechanism-based inactivators as probes of cytochrome P450 structure and function.

The cytochromes P450 superfamily of enzymes is a group of hemeproteins that catalyze the metabolism of an extensive series of compounds including drugs, chemical carcinogens, fatty acids, and steroids. They oxidize substrates ranging in size from ethylene to cyclosporin. Although significant efforts have been made to obtain structural information on the active sites of the microbial P450s, relatively little is currently known regarding the identities of the critical amino acid residues in the P450 active sites that are involved in substrate binding and catalysis. Since information on the crystal structures of the eukaryotic P450s has been relatively limited, investigators have used a variety of other techniques in attempts to elucide the structural features that play a role in the catalytic properties and substrate specificity at the enzyme active site. These include site-directed mutagenesis, natural mutations, homology modeling, mapping with aryl-iron complexes, affinity and photoaffinity labeling, and mechanism-based inactivators. A variety of different mechanism-based inactivators have proven to be useful in identifiying active site amino acid residues involved in substrate binding and catalysis. In this review we present a sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins. Labeled peptides isolated from the inactivated proteins have been analyzed by N-terminal amino acid sequencing in conjunction with mass spectrometry to determine the sites of covalent modification. Mechanistic studies aimed at identifying the basis for the inactivation following adduct formation are also presented.

Microsomal cytochrome P450 2C5: comparison to microbial P450s and unique features

Although microsomal P450s represent the majority of P450s, only microbial P450s have been amenable to crystal structure solution. We have recently solved the first crystal structure of a microsomal P450, 2C5, a progesterone hydroxylase from rabbit. We discuss the features of the protein in common with existing structures of microbial P450s and limitations of homology modeling mammalian P450s based on the microbial structures. Unique features involving membrane, substrate and cytochrome P450 reductase interactions are also discussed.

Cytochrome P450 proteins and potential utilization in biodegradation.

The cytochrome P450 enzymes are major catalysts involved in the oxidations of xenobiotic chemicals in microorganisms as well as higher animals and plants. Because of their functional roles, they offer potential in biodegradation technology. A number of microbial P450s have already been characterized and offer advantages in terms of their high catalytic rates and facile expression in microorganisms. One approach to extending the catalytic selectivity to more compounds in the environment is rational design. In three cases, the three-dimensional structures of bacterial cytochrome P450 enzymes are available and can be further understood through studies with molecular dynamics. Many mammalian cytochrome P450 enzymes have been studied extensively and have potential for biodegradation because of their broad catalytic selectivities (e.g., P450 2E1). Several advances have been made in the heterologous expression of these proteins in microorganisms. Improvements under development include electron transfer from flavodoxin and the use of cytochrome P450:NADPH-cytochrome P450 reductase fusion proteins. Random mutagenesis offers the potential of improving the catalytic activities of some of these proteins. Future challenges include the use of cytochrome P450 expression vectors in microorganisms capable of thriving in the environment; recent success in expression of vectors in Salmonella genotoxicity tester strains may be encouraging in this regard.