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

Budde, Michael; Maurer, Steffen; Schmid, Rolf D.; Urlacher, Vlada B.

doi:10.1007/s00253-004-1719-y

Cited by 55 publications

(24 citation statements)

References 28 publications

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“…Of the A-subfamily enzymes only CYP102A1, A2, A3, A5 and A7 have been studied in detail. The A2 and A3 enzymes show a preference for branched-chain over linear-chain fatty acids (Budde et al, 2004;Gustafsson et al, 2004;Lentz et al, 2004), A5 prefers polyunsaturated fatty acids over their saturated counterparts , while A7 is broadly similar to the A1 enzyme (Dietrich et al, 2008). The substrate binding and turnover kinetics of both A2 and A3 show sigmoidal behavior, indicating the binding of more than one substrate molecule and possible cooperativity.…”

Section: Introductionmentioning

confidence: 98%

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

et al. 2011

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Fatty acid binding and oxidation kinetics for wild type P450 BM3 (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k cat . The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450 BM3 could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.

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Section: Introductionmentioning

confidence: 98%

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

et al. 2011

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“…On the other hand, enzymes which are able to synthesize hydroxy LCFA can be found in nature. Cytochrome P450s, for example, are well known for their ability to break a C-H σ bond of fatty acid alkyl chain and attach a hydroxyl group to the carbon (Whitehouse et al 2012;Budde et al 2004;Lu et al 2010). Hydratases break a CC π bond in the middle of the alkyl chain and attach a hydroxyl group at one of the carbons (Joo et al 2012).…”

Section: Introductionmentioning

confidence: 99%

fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity

Bae

Park

Jung

et al. 2014

Appl Microbiol Biotechnol

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A major problem of long-chain fatty acid (LCFA) hydroxylation using Escherichia coli is that FadD (long-chain fatty acyl-CoA synthetase), which is necessary for exogenous LCFA transport, also initiates cellular consumption of LCFA. In this study, an effective method to prevent the cellular consumption of LCFA without impairing its transport is proposed. The main idea is that a heterologous enzyme which consumes LCFA can replace FadD in LCFA transport. For the model heterologous enzyme, CYP153A from Marinobacter aquaeolei, which converts palmitic acid into ω-hydroxy palmitic acid, was expressed in E. coli. When fadD was deleted from an E. coli strain, CYP153A indeed maintained the ability to transport LCFA. A disadvantage of fadD deletion mutant is the fact that FadD deficiency downregulates the transcription of fadL (outer membrane LCFA transporter) via FadR (fatty acid metabolism regulator protein), was solved by fadL overexpression from a plasmid. In addition, the overexpression of fadL was able to offset catabolite repression on fadL, allowing glucose to be used as the primary carbon source. In conclusion, the strain with fadD deletion and fadL overexpression showed 5.5-fold increase in productivity compared to the wild-type strain, converting 2.6 g/L (10.0 mM) of palmitic acid into 2.4 g/L (8.8 mM) of ω-hydroxy palmitic acid in a shake flask. This simple genetic manipulation can be applied to any LCFA hydroxylation using E. coli.

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“…[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%

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

Cited by 55 publications

References 28 publications

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

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