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Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity

ABSTRACT: Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.

SUBMITTER: Kokorin A

PROVIDER: S-EPMC8568981 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A tailor-made, self-sufficient and recyclable monooxygenase catalyst based on coimmobilized cytochrome P450 BM3 and glucose dehydrogenase.

Project description:Cytochrome P450 monooxygenases (P450s) promote hydroxylations in a broad variety of substrates. Their prowess in C-H bond functionalization renders P450s promising catalysts for organic synthesis. However, operating P450 reactions involve complex management of the main substrates, O2 and nicotinamide adenine dinucleotide phosphate (NAD(P)H) reducing equivalents against an overall background of low operational stability. Whole-cell biocatalysis, although often used, offers no general solution to these problems. Herein, we present the design of a tailor-made, self-sufficient, operationally stabilized and recyclable P450 catalyst on porous solid support. Using enzymes as fusion proteins with the polycationic binding module Zbasic2 , the P450s BM3 (from Bacillus megaterium) was coimmobilized with glucose dehydrogenase (type IV; from B. megaterium) on anionic sulfopropyl-activated carrier (ReliSorb SP). Immobilization via Zbasic2 enabled each enzyme to be loaded in controllable amount, thus maximizing the relative portion of the rate limiting P450 BM3 (up to 19.5?U/gcarrier ) in total enzyme immobilized. Using lauric acid as a representative P450 substrate that is poorly accessible to whole-cell catalysts, we demonstrate complete hydroxylation at low catalyst loading (?0.1?mol%) and efficient electron coupling (74%), inside of the catalyst particle, to the regeneration of NADPH from glucose (27 cycles) was achieved. The immobilized P450 BM3 showed a total turnover number of ?18,000, thus allowing active catalyst to be recycled up to 20 times. This study therefore supports the idea of practical heterogeneous catalysis by P450s systems immobilized on solid support.

| S-EPMC6836874 | biostudies-literature

Stabilization of the Reductase Domain in the Catalytically Self-Sufficient Cytochrome P450<sub>BM3</sub> by Consensus-Guided Mutagenesis.

Project description:The multidomain, catalytically self-sufficient cytochrome P450 BM-3 from Bacillus megaterium (P450BM3 ) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus-guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450BM3 . Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild-type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half-lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD-binding domain and that the optimal temperature (Topt ) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.

| S-EPMC5941085 | biostudies-literature

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles.

Project description:The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min-1; R255L: 1168 ± 163 min-1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.

| S-EPMC6651506 | biostudies-literature

Chimeric self-sufficient P450cam-RhFRed biocatalysts with broad substrate scope.

Project description:A high-throughput screening protocol for evaluating chimeric, self-sufficient P450 biocatalysts and their mutants against a panel of substrates was developed, leading to the identification of a number of novel biooxidation activities.

| S-EPMC3252848 | biostudies-literature

P450<sub>BM3</sub> fused to phosphite dehydrogenase allows phosphite-driven selective oxidations.

Project description:To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

| S-EPMC5320008 | biostudies-literature

Artificial Fusions between P450 BM3 and an Alcohol Dehydrogenase for Efficient (+)-Nootkatone Production.

Project description:Multi-enzyme cascades enable the production of valuable chemical compounds, and fusion of the enzymes that catalyze these reactions can improve the reaction outcome. In this work, P450 BM3 from Bacillus megaterium and an alcohol dehydrogenase from Sphingomonas yanoikuyae were fused to bifunctional constructs to enable cofactor regeneration and improve the in vitro two-step oxidation of (+)-valencene to (+)-nootkatone. An up to 1.5-fold increased activity of P450 BM3 was achieved with the fusion constructs compared to the individual enzyme. Conversion of (+)-valencene coupled to cofactor regeneration and performed in the presence of the solubilizing agent cyclodextrin resulted in up to 1080 mg L-1 (+)-nootkatone produced by the fusion constructs as opposed to 620 mg L-1 produced by a mixture of the separate enzymes. Thus, a two-step (+)-valencene oxidation was considerably improved through the simple method of enzyme fusion.

| S-EPMC9325546 | biostudies-literature

Photooxidation of cytochrome P450-BM3.

Project description:High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a Ru(II) photosensitizer to a nonnative cysteine near the heme (RuIIK97C-FeIIIP450), in order to promote electron transfer from the Fe(III) porphyrin to photogenerated Ru(III). The conjugate was structurally characterized by X-ray crystallography (2.4 ? resolution; Ru-Fe distance, 24 ?). Flash-quench oxidation of the ferric-aquo heme produces an Fe(IV)-hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.

| S-EPMC2973866 | biostudies-literature

Oleic acid based experimental evolution of Bacillus megaterium yielding an enhanced P450 BM3 variant.

Project description:BackgroundUnlike most other P450 cytochrome monooxygenases, CYP102A1 from Bacillus megaterium (BM3) is both soluble and fused to its redox partner forming a single polypeptide chain. Like other monooxygenases, it can catalyze the insertion of oxygen unto the carbon-hydrogen bond which can result in a wide variety of commercially relevant products for pharmaceutical and fine chemical industries. However, the instability of the enzyme holds back the implementation of a BM3-based biocatalytic industrial processes due to the important enzyme cost it would prompt.ResultsIn this work, we sought to enhance BM3's total specific product output by using experimental evolution, an approach not yet reported to improve this enzyme. By exploiting B. megaterium's own oleic acid metabolism, we pressed the evolution of a new variant of BM3, harbouring 34 new amino acid substitutions. The resulting variant, dubbed DE, increased the conversion of the substrate 10-pNCA to its product p-nitrophenolate 1.23 and 1.76-fold when using respectively NADPH or NADH as a cofactor, compared to wild type BM3.ConclusionsThis new DE variant, showed increased organic cosolvent tolerance, increased product output and increased versatility in the use of either nicotinamide cofactors NADPH and NADH. Experimental evolution can be used to evolve or to create libraries of evolved BM3 variants with increased productivity and cosolvent tolerance. Such libraries could in turn be used in bioinformatics to further evolve BM3 more precisely. The experimental evolution results also supports the hypothesis which surmises that one of the roles of BM3 in Bacillus megaterium is to protect it from exogenous unsaturated fatty acids by breaking them down.

| S-EPMC9281120 | biostudies-literature

Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes.

Project description:Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD+ , and NAD+ ) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD+ -dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.

| S-EPMC5299495 | biostudies-literature

An efficient light-driven P450 BM3 biocatalyst.

Project description:P450s are heme thiolate enzymes that catalyze the regio- and stereoselective functionalization of unactivated C-H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product/(mol enzyme/min).

| S-EPMC3938315 | biostudies-literature

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