A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

Appel, Daniel; Lutz-Wahl, Sabine; Fischer, Peter; Schwaneberg, Ulrich; Schmid, Rolf D.

doi:10.1016/s0168-1656(01)00249-8

Cited by 137 publications

(110 citation statements)

References 12 publications

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“…In order to demonstrate the general applicability of the biphasic system, octane and myristic acid -both substrates of CYP102A1 mutant A74G, F87V, L188Q with turnover rates of 1760 nmol(NADPH) nmol(P450) -1 min -1 [19] and 2100 nmol(NADPH) -1 nmol(P450) -1 min -1 , respectively -were also investigated.…”

Section: Conversion Of Octane and Myristic Acidmentioning

confidence: 99%

“…[13,14] Recently cloning and characterisation of two further fusion enzymes from Bacillus subtilis, which exhibit high homology to CYP102A1, was reported. [15][16][17] While a large number of publications describing novel evolved CYP102A1 mutants with altered substrate specificity exist, [18][19][20][21][22][23][24][25][26] there are only very few reports dealing with the use of these biocatalysts in preparative organic synthesis. [7,27] Beside strong doubts concerning the operational stability of the enzyme class, the high cost of the nicotinamide cofactor NADPH which has to be added in stochiometric or even higher amount (if uncoupling is taken into account) seems to make their in vitro application impractical.…”

Section: Introductionmentioning

confidence: 99%

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Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

et al. 2005

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Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP + -dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD + -dependent FDH was used.Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.

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Section: Conversion Of Octane and Myristic Acidmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

et al. 2005

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“…For screening mutant libraries of the P450 enzymes the monitoring of NAD(P)H consumption during substrate oxidation is usually used (Boddupalli et al 1990;Appel et al 2001;Lentz et al 2001). The modification of this method, so-called "alkali assay", was developed and adapted to the microtiterplate scale, and employed in high throughput screening (HTS) .…”

Section: Assays For P450 Monooxygenasesmentioning

confidence: 99%

“…A triple mutant (A74G/F87V/L188Q) was able to hydroxylate indole (Appel et al 2001) and also a wide range of other substrates that virtually do not have any resemblance to fatty acids which are regarded as the natural substrate of P450 BM-3. Thus, naphthalene (a polycyclic aromatic hydrocarbon), n-octane (an n-alkane), and 8-methylquinoline (a heteroarene) were oxidized.…”

Section: Protein Engineering Of P450 Monooxygenasesmentioning

confidence: 99%

Microbial P450 enzymes in biotechnology

Urlacher

Lutz-Wahl

Schmid

2004

Applied Microbiology and Biotechnology

200

100

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Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes, since they allow the introduction of oxygen even into non-activated carbon atoms in a sterically and optically selective manner which is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways which could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are covered in this review.3

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“…Activity and selectivity of bacterial CYPs can effectively be changed or/and improved by methods of site-directed mutagenesis or directed evolution (Appel et al 2001, Salazar et al 2003, Seng Wong et al 2004. Great efforts are presently being made to improve storage and operational stability of bacterial P450 enzymes (e.g.…”

Section: Introductionmentioning

confidence: 99%

Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis

Budde

Maurer

Schmid

et al. 2004

Appl Microbiol Biotechnol

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The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli. The recombinant enzyme formed was purified by immobilised metal chelat affinity chromatography (IMAC) and characterised. CYP102A2 is a 119 kDa self-sufficient monooxygenase, consisting of an FMN/FAD-containing reductase domain and a heme domain. The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from Bacillus megaterium (59%) and CYP102A3 from Bacillus subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 450 nm. It catalyses the oxidation of even-and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective ω-3, ω-2 and ω-1 hydroxylated fatty acids. Activity was highest towards oleic and linoleic acid (K M =17.4 ± 1.4 μM, k cat = 2244 ± 72 min -1 ), linoleic acid (K M =12.25 ± 1.8 μM, k cat = 1950 ± 84 min -1 ). Comparison of CYP102A2 homology model to CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in catalytic properties of these two enzymes.

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A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

Cited by 137 publications

References 12 publications

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Microbial P450 enzymes in biotechnology

Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis

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