Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis

Schroer, Kirsten; Kittelmann, Matthias; Lütz, Stephan

doi:10.1002/bit.22775

Cited by 91 publications

(65 citation statements)

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“…This progress can directly be of use for efficient and timesaving production of human drug metabolites. High yields and conversion rates can already be achieved by using corresponding human (Rushmore et al, 2000;Vail et al, 2005;Schroer et al, 2010;Geier et al, 2012;Schiffer et al, 2015) or suitable nonhuman (Taylor et al, 1999;Otey et al, 2006;Sawayama et al, 2009;Reinen et al, 2011;Di Nardo and Gilardi, 2012;Kiss et al, 2015;Ren et al, 2015) P450 enzymes in a whole-cell system to produce respective metabolites. The majority of published bacterial P450 enzymes used for the conversion of drugs are mutants of CYP102A1 (BM3) from Bacillus megaterium.…”

Section: Discussionmentioning

confidence: 99%

“…It has been shown that the utilization of human P450 enzymes enables the sufficient production of human drug metabolites employing baculovirus-infected insect cells expressing CYP3A4 or CYP2D9 (Rushmore et al, 2000), fission yeast expressing CYP2D6 (Peters et al, 2007) or CYP2C9 (Drǎgan et al, 2011;Neunzig et al, 2012), and Escherichia coli cells expressing CYP3A4, CYP2C9, and CYP1A2 (Vail et al, 2005). Since it is not mandatory to employ associated human P450 enzymes to synthesize human drug metabolites (Schroer et al, 2010;Geier et al, 2015), microbial, especially bacterial, P450 enzymes serve as a good alternative because they are convenient to handle and usually hold higher expression levels and activities, recommending the possibility to employ them as useful biocatalysts (Bernhardt, 2006). The genetic manipulation of bacterial P450 enzymes toward a drug metabolizing activity has been successfully demonstrated for several P450 enzymes including the most studied P450 102A1 (BM3), CYP102A1 (Whitehouse et al, 2012;Ren et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

Kern

Khatri

Litzenburger

et al. 2016

Drug Metabolism and Disposition

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The guidelines of the Food and Drug Administration and International Conference on Harmonization have highlighted the importance of drug metabolites in clinical trials. As a result, an authentic source for their production is of great interest, both for their potential application as analytical standards and for required toxicological testing. Since we have previously shown promising biotechnological potential of cytochromes P450 from the soil bacterium Sorangium cellulosum So ce56, herein we investigated the CYP267 family and its application for the conversion of commercially available drugs including nonsteroidal anti-inflammatory, antitumor, and antihypotensive drugs. The CYP267 family, especially CYP267B1, revealed the interesting ability to convert a broad range of substrates. We established substrate-dependent extraction protocols and also optimized the reaction conditions for the in vitro experiments and Escherichia coli-based whole-cell bioconversions. We were able to detect activity of CYP267A1 toward seven out of 22 drugs and the ability of CYP267B1 to convert 14 out of 22 drugs. Moderate to high conversions (up to 85% yield) were observed in our established whole-cell system using CYP267B1 and expressing the autologous redox partners, ferredoxin 8 and ferredoxin-NADP + reductase B. With our existing setup, we present a system capable of producing reasonable quantities of the human drug metabolites 49-hydroxydiclofenac, 2-hydroxyibuprofen, and omeprazole sulfone. Due to the great potential of converting a broad range of substrates, wild-type CYP267B1 offers a wide scope for the screening of further substrates, which will draw further attention to future biotechnological usage of CYP267B1 from S. cellulosum So ce56.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

Kern

Khatri

Litzenburger

et al. 2016

Drug Metabolism and Disposition

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“…Cytochromes P450 (CYPs) (EC 1.14.14.1) are a large super family of heme-containing monooxygenases that catalyzes mostly C-H hydroxylations on a wide range of endogenous as well as exogenous compounds [48]. Many of these chemical transformations are difficult to perform through synthetic methods, and therefore are of great interest for biocatalytic applications [49].…”

Section: Exploring Complex Enzymatic Mechanisms: P450 Monooxygenase Amentioning

confidence: 99%

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

Saa

Nielsen

2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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BackgroundAnalysis of limiting steps within enzyme-catalyzed reactions is fundamental to understand their behavior and regulation. Methods capable of unravelling control properties and exploring kinetic capabilities of enzymatic reactions would be particularly useful for protein and metabolic engineering. While single-enzyme control analysis formalism has previously been applied to well-studied enzymatic mechanisms, broader application of formalism is limited in practice by the limited amount of kinetic data and the difficulty of describing complex allosteric mechanisms. MethodsTo overcome these limitations, we present here a probabilistic framework enabling control analysis of previously unexplored mechanisms under uncertainty. By combining a thermodynamically consistent parameterization with an efficient Sequential Monte Carlosampler embedded in a Bayesian setting, this framework yields insights into the capabilities of enzyme-catalyzed reactions with modest kinetic information, provided that the catalytic mechanism and a thermodynamic reference point are defined. ResultsThe framework was used to unravel the impact of thermodynamic affinity, substrate saturation levels and effector concentrations on the flux control and response coefficients of a diverse set of enzymatic reactions. ConclusionsOur results highlight the importance of the metabolic context in the control analysis of isolated enzymes as well as the use of statistically sound methods for their interpretation. General SignificanceThis framework significantly expands our current capabilities for unravelling the control properties of general reaction kinetics with limited amount of information. This framework will be useful for both theoreticians and experimentalists in the field.

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“…Phase I metabolism involves the introduction or exposure of a reactive polar group on the xenobiotic via oxidation, resulting in a more reactive/water soluble metabolite to facilitate excretion and/or the induction of phase II metabolism. The cytochrome p450 super family of oxidases catalyse the majority of these reactions, although other oxidases, esterases, amidases, and monooxygenases can also be involved (Schroer et al, 2010). Phase II metabolism involves the conjugation of charged species such as glutathione, sulphate, glycine or glucuronic acid to the phase I metabolite to increase its water solubility (Kohalmy and Vrzal, 2011).…”

Section: Bioactivationmentioning

confidence: 99%

All Your Eggs in One Basket: Mechanisms of Xenobiotic Induced Female Reproductive Senescence

Sobinoff¹,

Bernstein²,

McLaughlin³

2012

Senescence

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Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis

Cited by 91 publications

References 32 publications

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

All Your Eggs in One Basket: Mechanisms of Xenobiotic Induced Female Reproductive Senescence

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