In vitro characterization of an enzymatic redox cascade composed of an alcohol dehydrogenase, an enoate reductases and a Baeyer-Villiger monooxygenase.
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ABSTRACT:
SUBMITTER: Oberleitner N
PROVIDER: S-EPMC4263279 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Despite the remarkable versatility displayed by flavin-dependent monooxygenases (FMOs) in natural product biosynthesis, one notably missing activity is the oxidative generation of carbonate functional groups. We describe a multifunctional Baeyer-Villiger monooxygenase, CcsB, which catalyzes the formation of an in-line carbonate in the macrocyclic portion of cytochalasin E. This study expands the repertoire of activities of FMOs and provides a possible synthetic strategy for transformation of ketones into carbonates.
| S-EPMC4062580 | biostudies-other
Project description:Flavin-containing Baeyer-Villiger monooxygenases employ NADPH and molecular oxygen to catalyze the insertion of an oxygen atom into a carbon-carbon bond of a carbonylic substrate. These enzymes can potentially be exploited in a variety of biocatalytic applications given the wide use of Baeyer-Villiger reactions in synthetic organic chemistry. The catalytic activity of these enzymes involves the formation of two crucial intermediates: a flavin peroxide generated by the reaction of the reduced flavin with molecular oxygen and the "Criegee" intermediate resulting from the attack of the flavin peroxide onto the substrate that is being oxygenated. The crystal structure of phenylacetone monooxygenase, a Baeyer-Villiger monooxygenase from the thermophilic bacterium Thermobifida fusca, exhibits a two-domain architecture resembling that of the disulfide oxidoreductases. The active site is located in a cleft at the domain interface. An arginine residue lays above the flavin ring in a position suited to stabilize the negatively charged flavin-peroxide and Criegee intermediates. This amino acid residue is predicted to exist in two positions; the "IN" position found in the crystal structure and an "OUT" position that allows NADPH to approach the flavin to reduce the cofactor. Domain rotations are proposed to bring about the conformational changes involved in catalysis. The structural studies highlight the functional complexity of this class of flavoenzymes, which coordinate the binding of three substrates (molecular oxygen, NADPH, and phenylacetone) in proximity of the flavin cofactor with formation of two distinct catalytic intermediates.
| S-EPMC516541 | biostudies-literature
Project description:Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the "in" position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the "variable" loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs.
| S-EPMC4966971 | biostudies-literature
Project description:Using highly purified enzyme preparations throughout, initial kinetic studies demonstrated that the isoenzymic 2,5- and 3,6-diketocamphane mono-oxygenases from Pseudomonas putida ATCC 17453 and the LuxAB luciferase from Vibrio fischeri ATCC 7744 exhibit commonality in being FMN-dependent two-component monooxygenases that promote redox coupling by the transfer of flavin reductase-generated FMNH2 by rapid free diffusion. Subsequent studies confirmed the comprehensive inter-species compatibility of both native and non-native flavin reductases with each of the tested monooxygenases. For all three monooxygenases, non-native flavin reductases from Escherichia coli ATCC 11105 and Aminobacter aminovorans ATCC 29600 were confirmed to be more efficient donators of FMNH2 than the corresponding tested native flavin reductases. Some potential practical implications of these outcomes are considered for optimising FMNH2-dependent biooxygenations of recognised practical and commercial value.
| S-EPMC9864536 | biostudies-literature
Project description:The molecular basis of allosteric effects, known to be caused by an effector docking to an enzyme at a site distal from the binding pocket, has been studied recently by applying directed evolution. Here, we utilize laboratory evolution in a different way, namely to induce allostery by introducing appropriate distal mutations that cause domain movements with concomitant reshaping of the binding pocket in the absence of an effector. To test this concept, the thermostable Baeyer-Villiger monooxygenase, phenylacetone monooxygenase (PAMO), was chosen as the enzyme to be employed in asymmetric Baeyer-Villiger reactions of substrates that are not accepted by the wild type. By using the known X-ray structure of PAMO, a decision was made regarding an appropriate site at which saturation mutagenesis is most likely to generate mutants capable of inducing allostery without any effector compound being present. After screening only 400 transformants, a double mutant was discovered that catalyzes the asymmetric oxidative kinetic resolution of a set of structurally different 2-substituted cyclohexanone derivatives as well as the desymmetrization of three different 4-substituted cyclohexanones, all with high enantioselectivity. Molecular dynamics (MD) simulations and covariance maps unveiled the origin of increased substrate scope as being due to allostery. Large domain movements occur that expose and reshape the binding pocket. This type of focused library production, aimed at inducing significant allosteric effects, is a viable alternative to traditional approaches to "designed" directed evolution that address the binding site directly.
| S-EPMC2840326 | biostudies-literature
Project description:Stenotrophomonas maltophilia PML168 was isolated from Wembury Beach on the English Coast from a rock pool following growth and selection on agar plates. Here we present the permanent draft genome sequence, which has allowed prediction of function for several genes encoding enzymes relevant to industrial biotechnology, including a novel flavoprotein monooxygenase.
| S-EPMC3415523 | biostudies-other
Project description:Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.
| S-EPMC3190734 | biostudies-literature
Project description:Baeyer-Villiger monooxygenases (BVMOs) are an emerging class of promising biocatalysts for the oxidation of ketones to prepare corresponding esters or lactones. Although many BVMOs have been reported, the development of highly efficient enzymes for use in industrial applications is desirable. In this work, we identified a BVMO from Rhodococcus pyridinivorans (BVMORp) with a high affinity toward aliphatic methyl ketones (Km < 3.0 μM). The enzyme was highly soluble and relatively stable, with a half-life of 23 h at 30°C and pH 7.5. The most effective substrate discovered so far is 2-hexanone (kcat = 2.1 s-1; Km = 1.5 μM). Furthermore, BVMORp exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMORp as the catalyst, a high concentration (26.0 g/liter; 200 mM) of methyl levulinate was completely converted to methyl 3-acetoxypropionate after 4 h, with a space-time yield of 5.4 g liter-1 h-1 Thus, BVMORp is a promising biocatalyst for the synthesis of 3-hydroxypropionate from readily available biobased levulinate to replace the conventional fermentation.IMPORTANCE BVMOs are emerging as a green alternative to traditional oxidants in the BV oxidation of ketones. Although many BVMOs are discovered and used in organic synthesis, few are really applied in industry, especially in the case of aliphatic ketones. Herein, a highly soluble and relatively stable monooxygenase from Rhodococcus pyridinivorans (BVMORp) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMORp possesses unusually high substrate loading during the catalysis of the oxidation of biobased methyl levulinate to 3-hydroxypropionic acid derivatives. This study indicates that the synthesis of 3-hydroxypropionate from readily available biobased levulinate by BVMORp-catalyzed oxidation holds great promise to replace traditional fermentation.
| S-EPMC6532031 | biostudies-literature
Project description:Baeyer-Villiger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa. This protein is phylogenetically distant from other well-characterized BVMOs. In order to study this new enzyme, we cloned its gene, expressed it in Escherichia coli and characterized the substrate scope of the Baeyer-Villiger monooxygenase from L. biflexa as a whole-cell biocatalyst. For this purpose, we performed the screening of a collection of ketones with variable structures and sizes, namely acyclic ketones, aromatic ketones, cyclic ketones, and fused ketones. As a result, we observed that this biocatalyst readily oxidized linear- and branched- medium-chain ketones, alkyl levulinates and linear ketones with aromatic substituents with excellent regioselectivity. In addition, this enzyme catalyzed the oxidation of 2-substituted cycloketone derivatives but showed an unusual selection against substituents in positions 3 or 4 of the ring.
| S-EPMC5407406 | biostudies-literature
Project description:BackgroundIt is widely accepted that the poor thermostability of Baeyer-Villiger monooxygenases limits their use as biocatalysts for applied biocatalysis in industrial applications. The goal of this study was to investigate the biocatalytic oxidation of 3,3,5-trimethylcyclohexanone using a thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) for the synthesis of branched ϵ-caprolactone derivatives as building blocks for tuned polymeric backbones. In this multi-enzymatic reaction, the thermostable cyclohexanone monooxygenase was fused to a phosphite dehydrogenase (PTDH) in order to ensure co-factor regeneration.ResultsUsing reaction engineering, the reaction rate and product formation of the regio-isomeric branched lactones were improved and the use of co-solvents and the initial substrate load were investigated. Substrate inhibition and poor product solubility were overcome using continuous substrate feeding regimes, as well as a biphasic reaction system with toluene as water-immiscible organic solvent. A maximum volumetric productivity, or space-time-yield, of 1.20 g L-1 h-1 was achieved with continuous feeding of substrate using methanol as co-solvent, while a maximum product concentration of 11.6 g L-1 was achieved with toluene acting as a second phase and substrate reservoir.ConclusionThese improvements in key process metrics therefore demonstrate progress towards the up-scaled Baeyer-Villiger monooxygenase-biocatalyzed synthesis of the target building blocks for polymer application. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
| S-EPMC6055809 | biostudies-literature