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Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications.
Li Z, Jiang Y, Guengerich FP, Ma L, Li S, Zhang W
(2020) J Biol Chem 295: 833-849
MeSH Terms: Biocatalysis, Biotechnology, Cytochrome P-450 Enzyme System, Metabolic Engineering, Protein Engineering, Steroids, Substrate Specificity, Xenobiotics
Show Abstract · Added March 3, 2020
Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C-H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.
© 2020 Li et al.
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8 MeSH Terms
A Role for the Orphan Human Cytochrome P450 2S1 in Polyunsaturated Fatty Acid -1 Hydroxylation Using an Untargeted Metabolomic Approach.
Fekry MI, Xiao Y, Berg JZ, Guengerich FP
(2019) Drug Metab Dispos 47: 1325-1332
MeSH Terms: Animals, Cytochrome P-450 CYP4A, Cytochrome P-450 Enzyme System, Fatty Acids, Unsaturated, Female, Gastrointestinal Tract, Humans, Hydroxylation, Isotope Labeling, Metabolomics, Rats
Show Abstract · Added March 3, 2020
Cytochrome P450 (P450) 2S1 is one of the orphan P450s, known to be expressed but not having a defined function with an endogenous substrate or in drug oxidations. Although it has been clearly demonstrated to catalyze reductive reactions, its role in NADPH-dependent oxidations has been ambiguous. In our efforts to characterize orphan human P450 enzymes, we used an untargeted liquid chromatography-mass spectromterymetabolomic approach with recombinant human P450 2S1 and extracts of rat stomach and intestine, sites of P450 2S1 localization in humans and animals. The search yielded several candidates, including the product 19-hydroxyarachidonic acid. Subsequent O analysis and in vitro studies with commercial arachidonic acid and 19-hydroxyarachidonic acid were used to validate -1 hydroxylation of the former molecule as a NADPH- and O-dependent reaction. Steady-state kinetic assays were done for -1 hydroxylation reactions of P450 2S1 with several other long-chain fatty acids, including arachidonic, linoleic, -linolenic, eicosapentaenoic, and docosapentaenoic acids. Rates of hydroxylation were slow, but no detectable activity was seen with either medium-chain length or saturated fatty acids. P450 2S1 is known to be expressed, at least at the mRNA level, to the extent of some other non-3A subfamily P450s in the human gastrointestinal tract, and the activity may be relevant. We conclude that P450 2S1 is a fatty acid -1 hydroxylase, although the physiologic relevance of these oxidations remains to be established. The metabolomic approaches we employed in this study are feasible for orphan P450s and other enzymes, in regard to annotation of function, in mammals and other organisms. SIGNIFICANCE STATEMENT: An untargeted mass spectrometry approach was utilized to identify -1 hydroxylation of arachidonic acid as an oxidative reaction catalyzed by human cytochrome P450 2S1. The enzyme also catalyzes the relatively slow -1 hydroxylation of several other unsaturated long-chain fatty acids.
Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.
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11 MeSH Terms
Kinetic Modeling of Steady-State Situations in Cytochrome P450 Enzyme Reactions.
Guengerich FP
(2019) Drug Metab Dispos 47: 1232-1239
MeSH Terms: Computer Simulation, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, Humans, Kinetics, Ligands
Show Abstract · Added March 3, 2020
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, ), and the effect of the order is more pronounced with a stronger inhibitor. Substrate depletion alters parameters (Michaelis constant, ) and can generate apparently sigmoidal plots. A rate-limiting step after product formation lowers the apparent and distorts Consumption of an inhibitor during a reaction affects 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.
Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.
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6 MeSH Terms
Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes.
Guengerich FP, Wilkey CJ, Phan TTN
(2019) J Biol Chem 294: 10928-10941
MeSH Terms: Catalysis, Cytochrome P-450 CYP2D6, Cytochrome P-450 CYP2E1, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, Humans, Kinetics, Lauric Acids, Ligands, Molecular Conformation, Oxidation-Reduction, Palmitic Acid, Protein Binding, Protein Conformation, Spiro Compounds, Substrate Specificity
Show Abstract · Added March 3, 2020
Cytochrome P450 (P450) enzymes are major catalysts involved in the oxidations of most drugs, steroids, carcinogens, fat-soluble vitamins, and natural products. The binding of substrates to some of the 57 human P450s and other mammalian P450s is more complex than a two-state system and has been proposed to involve mechanisms such as multiple ligand occupancy, induced-fit, and conformational-selection. Here, we used kinetic analysis of binding with multiple concentrations of substrates and computational modeling of these data to discern possible binding modes of several human P450s. We observed that P450 2D6 binds its ligand rolapitant in a mechanism involving conformational-selection. P450 4A11 bound the substrate lauric acid via conformational-selection, as did P450 2C8 with palmitic acid. Binding of the steroid progesterone to P450 21A2 was also best described by a conformational-selection model. Hexyl isonicotinate binding to P450 2E1 could be described by either a conformational-selection or an induced-fit model. Simulation of the binding of the ligands midazolam, bromocriptine, testosterone, and ketoconazole to P450 3A4 was consistent with an induced-fit or a conformational-selection model, but the concentration dependence of binding rates for varying both P450 3A4 and midazolam concentrations revealed discordance in the parameters, indicative of conformational-selection. Binding of the P450s 2C8, 2D6, 3A4, 4A11, and 21A2 was best described by conformational-selection, and P450 2E1 appeared to fit either mode. These findings highlight the complexity of human P450-substrate interactions and that conformational-selection is a dominant feature of many of these interactions.
© 2019 Guengerich et al.
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17 MeSH Terms
Oxidation of Flavone, 5-Hydroxyflavone, and 5,7-Dihydroxyflavone to Mono-, Di-, and Tri-Hydroxyflavones by Human Cytochrome P450 Enzymes.
Nagayoshi H, Murayama N, Kakimoto K, Tsujino M, Takenaka S, Katahira J, Lim YR, Kim D, Yamazaki H, Komori M, Guengerich FP, Shimada T
(2019) Chem Res Toxicol 32: 1268-1280
MeSH Terms: Cytochrome P-450 Enzyme System, Flavones, Flavonoids, Humans, Molecular Structure, Oxidation-Reduction
Show Abstract · Added March 3, 2020
Biologically active plant flavonoids, including 5,7-dihydroxyflavone (57diOHF, chrysin), 4',5,7-trihydroxyflavone (4'57triOHF, apigenin), and 5,6,7-trihydroxyflavone (567triOHF, baicalein), have important pharmacological and toxicological significance, e.g., antiallergic, anti-inflammatory, antioxidative, antimicrobial, and antitumorgenic properties. In order to better understand the metabolism of these flavonoids in humans, we examined the oxidation of flavone, 5-hydroxyflavone (5OHF), and 57diOHF to various products by human cytochrome P450 (P450 or CYP) and liver microsomal enzymes. Individual human P450s and liver microsomes oxidized flavone to 6-hydroxyflavone, small amounts of 5OHF, and 11 other monohydroxylated products at different rates and also produced several dihydroxylated products (including 57diOHF and 7,8-dihydroxyflavone) from flavone. We also found that 5OHF was oxidized by several P450 enzymes and human liver microsomes to 57diOHF and further to 567triOHF, but the turnover rates in these reactions were low. Interestingly, both CYP1B1.1 and 1B1.3 converted 57diOHF to 567triOHF at turnover rates (on the basis of P450 contents) of >3.0 min, and CYP1A1 and 1A2 produced 567triOHF at rates of 0.51 and 0.72 min, respectively. CYP2A13 and 2A6 catalyzed the oxidation of 57diOHF to 4'57triOHF at rates of 0.7 and 0.1 min, respectively. Our present results show that different P450s have individual roles in oxidizing these phytochemical flavonoids and that these reactions may cause changes in their biological and toxicological properties in mammals.
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6 MeSH Terms
In vitro safety pharmacology evaluation of 2-hydroxybenzylamine acetate.
Fuller JC, Pitchford LM, Morrison RD, Daniels JS, Flynn CR, Abumrad NN, Oates JA, Boutaud O, Rathmacher JA
(2018) Food Chem Toxicol 121: 541-548
MeSH Terms: Adult, Benzylamines, Blood Proteins, Cytochrome P-450 Enzyme System, ERG1 Potassium Channel, Erythrocytes, Female, Gene Expression Regulation, Enzymologic, Hepatocytes, Humans, Male, Middle Aged, Mutagenicity Tests, Salmonella typhimurium
Show Abstract · Added April 15, 2019
2-hydroxybenzylamine (2-HOBA), a compound found in buckwheat, is a potent scavenger of reactive γ-ketoaldehydes, which are increased in diseases associated with inflammation and oxidative stress. While the potential of 2-HOBA is promising, studies were needed to characterize the safety of the compound before clinical trials. In a series of experiments, the risks of 2-HOBA-mediated mutagenicity and cardio-toxicity were assessed in vitro. The effects of 2-HOBA on the mRNA expression of select cytochrome P450 (CYP) enzymes were also assessed in cryopreserved human hepatocytes. Further, the distribution and metabolism of 2-HOBA in blood were determined. Our results indicate that 2-HOBA is not cytotoxic or mutagenic in vitro and does not induce the expression of CYP1A2, CYP2B6, or CYP3A4 in human hepatocytes. The results of the hERG testing showed a low risk of cardiac QT wave prolongation. Plasma protein binding and red blood cell distribution characteristics indicate low protein binding and no preferential distribution into erythrocytes. The major metabolites identified were salicylic acid and the glycoside conjugate of 2-HOBA. Together, these findings support development of 2-HOBA as a nutritional supplement and provide important information for the design of further preclinical safety studies in animals as well as for human clinical trials with 2-HOBA.
Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.
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MeSH Terms
The Cytochrome P450 Slow Metabolizers CYP2C9*2 and CYP2C9*3 Directly Regulate Tumorigenesis via Reduced Epoxyeicosatrienoic Acid Production.
Sausville LN, Gangadhariah MH, Chiusa M, Mei S, Wei S, Zent R, Luther JM, Shuey MM, Capdevila JH, Falck JR, Guengerich FP, Williams SM, Pozzi A
(2018) Cancer Res 78: 4865-4877
MeSH Terms: Animals, Arachidonic Acid, Arachidonic Acids, Carcinogenesis, Carcinoma, Non-Small-Cell Lung, Cell Line, Tumor, Cytochrome P-450 CYP2C9, Cytochrome P-450 Enzyme System, Eicosanoids, Endothelial Cells, Humans, Mice, Polymorphism, Single Nucleotide, Xenograft Model Antitumor Assays
Show Abstract · Added October 25, 2018
Increased expression of cytochrome P450 CYP2C9, together with elevated levels of its products epoxyeicosatrienoic acids (EET), is associated with aggressiveness in cancer. Cytochrome P450 variants and encode proteins with reduced enzymatic activity, and individuals carrying these variants metabolize drugs more slowly than individuals with wild-type , potentially affecting their response to drugs and altering their risk of disease. Although genetic differences in CYP2C9-dependent oxidation of arachidonic acid (AA) have been reported, the roles of CYP2C9*2 and CYP2C9*3 in EET biosynthesis and their relevance to disease are unknown. Here, we report that CYP2C9*2 and CYP2C9*3 metabolize AA less efficiently than CYP2C9*1 and that they play a role in the progression of non-small cell lung cancer (NSCLC) via impaired EET biosynthesis. When injected into mice, NSCLC cells expressing CYP2C9*2 and CYP2C9*3 produced lower levels of EETs and developed fewer, smaller, and less vascularized tumors than cells expressing CYP2C9*1. Moreover, endothelial cells expressing these two variants proliferated and migrated less than cells expressing CYP2C*1. Purified CYP2C9*2 and CYP2C9*3 exhibited attenuated catalytic efficiency in producing EETs, primarily due to impaired reduction of these two variants by NADPH-P450 reductase. Loss-of-function SNPs within and were associated with improved survival in female cases of NSCLC. Thus, decreased EET biosynthesis represents a novel mechanism whereby CYPC29*2 and CYP2C9*3 exert a direct protective role in NSCLC development. These findings report single nucleotide polymorphisms in the human CYP2C9 genes, and , exert a direct protective role in tumorigenesis by impairing EET biosynthesis. .
©2018 American Association for Cancer Research.
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14 MeSH Terms
Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration.
Albertolle ME, Phan TTN, Pozzi A, Guengerich FP
(2018) Mol Cell Proteomics 17: 889-900
MeSH Terms: Animals, Biocatalysis, Cysteine, Cytochrome P-450 Enzyme System, Humans, Hydrogen Peroxide, Kidney, Mice, Transgenic, Microsomes, Liver, Oxidation-Reduction, Pharmaceutical Preparations, Recombinant Proteins, Staining and Labeling, Sulfenic Acids, Sulfhydryl Compounds
Show Abstract · Added March 14, 2018
The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. (2017) 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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15 MeSH Terms
Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone.
Kakimoto K, Murayama N, Takenaka S, Nagayoshi H, Lim YR, Kim V, Kim D, Yamazaki H, Komori M, Guengerich FP, Shimada T
(2019) Xenobiotica 49: 131-142
MeSH Terms: Chromatography, Liquid, Cytochrome P-450 CYP2A6, Cytochrome P-450 Enzyme System, Flavanones, Flavones, Humans, Kinetics, Mass Spectrometry, Molecular Docking Simulation, Oxidation-Reduction
Show Abstract · Added March 14, 2018
1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2'- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min and 1.3 min, respectively. Other flavanone metabolites were 4'-, 3'- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed.
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10 MeSH Terms
Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes.
Gonzalez E, Johnson KM, Pallan PS, Phan TTN, Zhang W, Lei L, Wawrzak Z, Yoshimoto FK, Egli M, Guengerich FP
(2018) J Biol Chem 293: 541-556
MeSH Terms: Animals, Cytochrome P-450 Enzyme System, Humans, Hydroxysteroids, Protein Conformation, Steroid 17-alpha-Hydroxylase, Zebrafish
Show Abstract · Added March 14, 2018
Cytochrome P450 (P450) 17A1 catalyzes the oxidations of progesterone and pregnenolone and is the major source of androgens. The enzyme catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction, and several mechanisms have been proposed for the latter step. Zebrafish P450 17A2 catalyzes only the 17α-hydroxylations. We previously reported high similarity of the crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1. Five residues near the heme, which differed, were changed. We also crystallized this five-residue zebrafish P450 17A1 mutant, and the active site still resembled the structure in the other proteins, with some important differences. These P450 17A1 and 17A2 mutants had catalytic profiles more similar to each other than did the wildtype proteins. Docking with these structures can explain several minor products, which require multiple enzyme conformations. The 17α-hydroperoxy (OOH) derivatives of the steroids were used as oxygen surrogates. Human P450 17A1 and zebrafish P450s 17A1 and P450 17A2 readily converted these to the lyase products in the absence of other proteins or cofactors (with catalytically competent kinetics) plus hydroxylated 17α-hydroxysteroids. The 17α-OOH results indicate that a "Compound I" (FeO) intermediate is capable of formation and can be used to rationalize the products. We conclude that zebrafish P450 17A2 is capable of lyase activity with the 17α-OOH steroids because it can achieve an appropriate conformation for lyase catalysis in this system that is precluded in the conventional reaction.
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7 MeSH Terms