Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio‐, Stereo‐ and Chemoselectivity

Seifert, Alexander; Vomund, Sandra; Grohmann, Katrin; Kriening, Sebastian; Urlacher, Vlada B.; Laschat, Sabine; Pleiss, Jürgen

doi:10.1002/cbic.200990033

Cited by 9 publications

(11 citation statements)

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“…Engineered cytochrome P450s from Pseudomonas putida and Bacillus megaterium were shown to convert valencene into nootkatone [8,9] muticus [10] was shown to catalyse mono-oxygenation of (+)-valencene to nootkatol. Mutations in substrate recognition regions of this enzyme further improved the catalytic efficiency of nootkatol formation [10].…”

Section: Introductionmentioning

confidence: 99%

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

et al. 2010

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a b s t r a c tChicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.

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Section: Introductionmentioning

confidence: 99%

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

et al. 2010

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“…Rational design and directed evolution have already been successfully used, both separately and in tandem, to generate BM3 mutants that are capable of metabolizing both native and exogenous substrates with increased activities and altered regio-and stereoselectivities. These substrates include aromatics (Carmichael and Wong, 2001;Li et al, 2001a;Whitehouse et al, 2008), alkanes (Appel et al, 2001;Peters et al, 2003;Kubo et al, 2006), hydrocarbons (Seifert et al, 2009), and carboxylic acids (Munzer et al, 2005). Moreover, van Vugt-Lussenburg et al (2007) and others (Landwehr et al, 2006;Otey et al, 2006;Kim et al, 2008Kim et al, , 2009Kim et al, , 2011Rentmeister et al, 2009;Sawayama et al, 2009) have shown that P450 BM3 mutants can also be used for the production of both human relevant and BM3 unique drug metabolites.…”

Section: Introductionmentioning

confidence: 99%

Efficient Screening of Cytochrome P450 BM3 Mutants for Their Metabolic Activity and Diversity toward a Wide Set of Drug-Like Molecules in Chemical Space

Reinen¹,

Leeuwen²,

Li³

et al. 2011

Drug Metabolism and Disposition

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ABSTRACT:In the present study, the diversity of a library of drug-metabolizing bacterial cytochrome P450 (P450) BM3 mutants was evaluated by a liquid chromatography-mass spectrometry (LC-MS)-based screening method. A strategy was designed to identify a minimal set of BM3 mutants that displays differences in regio-and stereoselectivities and is suitable to metabolize a large fraction of drug chemistry space. We first screened the activities of six structurally diverse BM3 mutants toward a library of 43 marketed drugs (encompassing a wide range of human P450 phenotypes, cLogP values, charges, and molecular weights) using a rapid LC-MS method with an automated method development and data-processing system. Significant differences in metabolic activity were found for the mutants tested and based on this drug library screen; nine structurally diverse probe drugs were selected that were subsequently used to study the metabolism of a library of 14 BM3 mutants in more detail. Using this alternative screening strategy, we were able to select a minimal set of BM3 mutants with high metabolic activities and diversity with respect to substrate specificity and regiospecificity that could produce both human relevant and BM3 unique drug metabolites. This panel of four mutants (M02, MT35, MT38, and MT43) was capable of producing P450-mediated metabolites for 41 of the 43 drugs tested while metabolizing 77% of the drugs by more than 20%. We observed this as the first step in our approach to use of bacterial P450 enzymes as general reagents for lead diversification in the drug development process and the biosynthesis of drug(-like) metabolites.

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“…[78][79][80][81] The resulting products (alcohols, ketones, and epoxides) constitute important flavor and fragrance compounds, pheromones, or natural drugs. As an example, (þ)-nootkatone 51 constitutes a high-value sesquiterpene used in the flavor and fragrance industry with a market price of 4,000 to 6,500 D / kg.…”

Section: Cytochrome P450 Monooxygenasesmentioning

confidence: 99%

Biocatalytic Asymmetric Oxidations in Stereoselective Synthesis

Schallmey

Marı́a

Bracco

2013

Stereoselective Synthesis of Drugs and Natural Products

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Enzyme‐catalyzed oxidations represent a versatile approach for the production of optically active building blocks and natural products. Moreover, enzymatic reaction conditions are often mild and with high atom economy, thus, enabling the basis for future environmentally friendly chemistry. As a result, several biocatalytic oxidative processes have already been implemented on an industrial scale as well, clearly showing that modern biocatalysis can compete economically with other chemical‐based processes. This chapter thoroughly describes the different types of oxidative enzymes that can be found in nature, namely, dehydrogenases, oxygenases, oxidases, and peroxidases, with emphasis on their mechanism and occurrence. Furthermore, several outstanding examples of biocatalytic applications of these enzymes are given—either by means of recombinant whole cells or isolated enzymes—involving the production of different drugs and natural products in which a catalytic step is catalyzed by these oxidative enzymes.

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Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio‐, Stereo‐ and Chemoselectivity

Cited by 9 publications

References 0 publications

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

Efficient Screening of Cytochrome P450 BM3 Mutants for Their Metabolic Activity and Diversity toward a Wide Set of Drug-Like Molecules in Chemical Space

Biocatalytic Asymmetric Oxidations in Stereoselective Synthesis

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