Preparation of human metabolites of propranolol using laboratory‐evolved bacterial cytochromes P450

Otey, Christopher R.; Bandara, Geethani; Lalonde, James J.; Takahashi, Katsuyuki; Arnold, Frances H.

doi:10.1002/bit.20744

Cited by 135 publications

(111 citation statements)

References 37 publications

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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%

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%

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 obvious route is to use isolated human P450s, but these complex multiprotein systems are membrane-bound, poorly stable, cofactor-dependent, and generally exhibit low reaction rates [7,8]. More promising results have been reported for laboratory-evolved bacterial P450s, which are capable of catalyzing the H 2 O 2 -dependent hydroxylation of pharmaceuticals via the "peroxide shunt" pathway [9]. Recent studies, however, have demonstrated that this approach needs further optimization [10].…”

Section: Introductionmentioning

confidence: 99%

Preparation of human drug metabolites using fungal peroxygenases

Poraj-Kobielska

Kinne

Ullrich

et al. 2011

Biochemical Pharmacology

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The synthesis of hydroxylated and O-or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

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“…In an effort to evolve the cyclization specificity of the Thermoanerobacterium thermosulfurigenes strain EM1 (Tabium) CGTase into R-amylase like hydrolytic activity on starch, we separately applied error-prone PCR (epPCR) and saturated mutagenesis, also evaluating the effectiveness of directed evolution techniques for interconversion of reaction specificities. Directed evolution has emerged as a powerful method for the creation of proteins with altered properties, e.g., tighter binding, resistance to denaturing conditions, and modified and/or improved reaction specificities (8)(9)(10)(11)(12)(13). Several directed evolution methods are commonly used nowadays, epPCR, DNA shuffling, site-saturation mutagenesis, etc., in the generation of genetic diversity, as reviewed (14,15).…”

mentioning

confidence: 99%

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

2007

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Glycoside hydrolase family 13 (GH13) members have evolved to possess various distinct reaction specificities despite the overall structural similarity. In this study we investigated the evolutionary input required to effeciently interchange these specificities and also compared the effectiveness of laboratory evolution techniques applied, i.e., error-prone PCR and saturation mutagenesis. Conversion of our model enzyme, cyclodextrin glucanotransferase (CGTase), into an R-amylase like hydrolytic enzyme by saturation mutagenesis close to the catalytic core yielded a triple mutant (A231V/F260W/F184Q) with the highest hydrolytic rate ever recorded for a CGTase, similar to that of a highly active R-amylase, while cyclodextrin production was virtually abolished. Screening of a much larger, error-prone PCR generated library yielded far less effective mutants. Our results demonstrate that it requires only three mutations to change CGTase reaction specificity into that of another GH13 enzyme. This suggests that GH13 members may have diversified by introduction of a limited number of mutations to the common ancestor, and that interconversion of reaction specificites may prove easier than previously thought.

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Preparation of human metabolites of propranolol using laboratory‐evolved bacterial cytochromes P450

Cited by 135 publications

References 37 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

Preparation of human drug metabolites using fungal peroxygenases

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

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