Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

Beier, Andy; Bordewick, Sven; Genz, Maika; Schmidt, Sandy; Bergh, Tom van den; Peters, Christin; Joosten, Han; Bornscheuer, Uwe T.

doi:10.1002/cbic.201600484

Cited by 47 publications

(51 citation statements)

References 34 publications

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“…[18] , ! [26,27] We measured rates of NADPH depletion using various additives andrates of H 2 O 2 formation via acolorimetrica ssay,a lways starting from 100 mMN ADPH (we determined K M as approx. [19] , ‰ ref.…”

Section: Resultsmentioning

confidence: 99%

Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation

et al. 2017

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This paper describest he stabilizationo f flavin-dependent monooxygenases under reaction conditions,u sing an engineered formulation of additives (the natural cofactors NADPH andF AD,a nd superoxide dismutase and catalase as catalytic antioxidants). This way,a10 3 -t o1 0 4 -foldi ncrease of the half-life was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. Thes tabilized enzymes are highly valuedf or theirs ynthetic potential in biotechnology and medicinalc hemistry (enantioselective sulfur, nitrogen and Baeyer-Villiger oxidations;o xidative human metabolism), but widespread application was so far hindered by their notoriousf ragility. Our technology immediately enables their use,d oes not require structural knowledge of the biocatalyst, and creates as trong basis for the targeted development of improvedvariants by mutagenesis.

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Section: Resultsmentioning

confidence: 99%

Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation

et al. 2017

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“…We were interested whether the concept can be extended towards the utilization of NADPH for selective oxyfunctionalisation reactions, and if side-reactions and physiological side-effects might be a problem for a catalytic application. In order to test the performance of the system, one of the most widely used Baeyer-Villiger monooxygenases (BVMO), the NADPH-specific cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus NCIMB 9871 was chosen as a model (Scheme 1) [2,14]. …”

Section: Introductionmentioning

confidence: 99%

Enzymatic Oxyfunctionalization Driven by Photosynthetic Water-Splitting in the Cyanobacterium Synechocystis sp. PCC 6803

et al. 2017

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Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts of reduced nicotinamide to a recombinant enoate reductase opened a new strategy for overcoming this hurdle. This paper presents the expansion of the photosynthetic regeneration system to a Baeyer-Villiger monooxygenase. Despite the potential of this strategy, this work also presents some of the encountered challenges as well as possible solutions, which will require further investigation. The successful enzymatic oxygenation shows that cyanobacterial whole-cell biocatalysis is an applicable approach that allows fuelling selective oxyfunctionalisation reactions at the expense of light and water. Yet, several hurdles such as side-reactions and the cell-density limitation, probably due to self-shading of the cells, will have to be overcome on the way to synthetic applications.

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“…The gene encoding HtBVMO was identified in plasmid pHTUR01. HtBVMO shows 50% sequence identity with PAMO and conserved regions containing a number of known structural and functional motifs: (i) two Rossmann fold motifs (GxGxxG/A), involved in dinucleotide cofactor binding [16], (ii) the aforementioned motif for Type I BVMOs, (iii) the [A/G]GxWxxxx[F/Y]P[G/M]xxxD and (iv) Dx[I/L][V/I]xxTG[Y/F] motifs, all identifying this sequence as a putative BVMO [15,17,18]. A Clustal Omega alignment of HtBVMO with PAMO and CHMO from Acinetobacter NCIMB 9871 is shown in supplementary Figure S1.…”

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confidence: 99%

Unique Features of a New Baeyer–Villiger Monooxygenase from a Halophilic Archaeon

et al. 2020

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Type I Baeyer–Villiger monooxygenases (BVMOs) are flavin-dependent monooxygenases that catalyze the oxidation of ketones to esters or lactones, a reaction otherwise performed in chemical processes by employing hazardous and toxic peracids. Even though various BVMOs are extensively studied for their promising role in industrial biotechnology, there is still a demand for enzymes that are able to retain activity at high saline concentrations. To this aim, and based on comparative in silico analyses, we cloned HtBVMO from the extremely halophilic archaeon Haloterrigena turkmenica DSM 5511. When expressed in standard mesophilic cell factories, proteins adapted to hypersaline environments often behave similarly to intrinsically disordered polypeptides. Nevertheless, we managed to express HtBVMO in Escherichia coli and could purify it as active enzyme. The enzyme was characterized in terms of its salt-dependent activity and resistance to some water–organic-solvent mixtures. Although HtBVMO does not seem suitable for industrial applications, it provides a peculiar example of an alkalophilic and halophilic BVMO characterized by an extremely negative charge. Insights into the behavior and structural properties of such salt-requiring may contribute to more efficient strategies for engineering the tuned stability and solubility of existing BVMOs.

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Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

Cited by 47 publications

References 34 publications

Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation

Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation

Enzymatic Oxyfunctionalization Driven by Photosynthetic Water-Splitting in the Cyanobacterium Synechocystis sp. PCC 6803

Unique Features of a New Baeyer–Villiger Monooxygenase from a Halophilic Archaeon

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