Hot spots for the protein engineering of Baeyer-Villiger monooxygenases

Balke, Kathleen; Beier, Andy; Bornscheuer, Uwe T.

doi:10.1016/j.biotechadv.2017.11.007

Cited by 75 publications

(60 citation statements)

References 121 publications

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“…The substrate scope displayed by these fascinating biocatalysts can be broad; even α‐substituted ketone substrates are accepted . Efficient cofactor regeneration systems to recycle nicotinamide adenine dinucleotide phosphate (NADP + ) back to NADPH exist …”

Section: Resultsmentioning

confidence: 99%

“…reported the synthesis of norcamphor lactone on 80 mmol scale, in 81% yield and 5 days reaction time which opened the door to further investigations to modulate the entire process in order to further scale it up to a better yield. We have previously reported that an engineered thermostable variant of CHMO Acineto , harboring four substitutions and referred to as CHMO Acineto _QM, was able to fully convert the nonnatural bulky substrate norcamphor (the major product is the normal lactone, 93.4%) and act on the more substituted substrate (−)‐ cis ‐verbanone . Scaling up this transformation from 2 to 33 mM of the substrate norcamphor in shake‐flask enabled the biosynthesis of norcamphor lactone in 0.5 g scale, stressing the high potential of the enzyme CHMO Acineto _QM to be used for the production of industrially relevant products (Scheme ).…”

Section: Resultsmentioning

confidence: 99%

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Lactone monomers obtained by enzyme catalysis and their use in reversible thermoresponsive networks

Farhat

Biundo

Stamm

et al. 2020

J of Applied Polymer Sci

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The cover image by Per-Olof Syrén and colleagues shows a green route to new star-shaped polymeric materials that can reconstruct themselves by means of Diels-Alder chemistry when stimulated by heat. The image shows how enzyme catalysis successfully afforded two key reaction steps. In addition, the image reveals the conversion of bulky bicyclic ketone to its corresponding activated lactone monomer, as well as end-capping of the chemically synthesized polymer with the renewable building block furoic acid.Volume 137, Number 18 was mailed the week of February 3, 2020.ARTICLES 48608 Hydrophobic-associated polymer-based laponite nanolayered silicate composite as filtrate reducer for water-based drilling fluid at high temperature

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Lactone monomers obtained by enzyme catalysis and their use in reversible thermoresponsive networks

Farhat

Biundo

Stamm

et al. 2020

J of Applied Polymer Sci

View full text Add to dashboard Cite

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“…With the exception of directed evolution studies influencing the substrate scope and selectivity/specificity through allosteric effects [22,23], protein engineering of BVMOs to improve substrate acceptance or regio-, enantio-, and stereospecificity/selectivity has mostly focused on "hot spot" residues lining the active site [4,5]. NADP(H), however, remains bound during the entire catalytic cycle of BVMOs, and early kinetic [24] and structural studies [25] on FMO have already revealed its dual function in this class of enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…However, their use as alternatives to chemical Baeyer-Villiger catalysts, and their ability to also catalyze heteroatom oxidation, has driven much of the research over the past two decades. This has led the search for new and improved BVMOs to expand on the current substrate-scope, enhanced regio-, enantio-and stereospecificity/selectivity, as well as stability of these promising biocatalysts [1,[4][5][6].…”

Section: Introductionmentioning

confidence: 99%

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

et al. 2020

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Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that primarily convert ketones to esters, but can also catalyze heteroatom oxidation. Several structural studies have highlighted the importance of the 'control loop' in BVMOs, which adopts different conformations during catalysis. Central to the 'control loop' is a conserved tryptophan that has been implicated in NADP(H) binding. BVMO AFL210 from Aspergillus flavus, however, contains a threonine in the equivalent position. Here, we report the structure of BVMO AFL210 in complex with NADP + in both the 'open' and 'closed' conformations. In neither conformation does Thr513 contact the NADP + . Although mutagenesis of Thr513 did not significantly alter the substrate scope, changes in peroxyflavin stability and reaction rates were observed. Mutation of this position also brought about changes in the regioand enantioselectivity of the enzyme. Moreover, lower rates of overoxidation during sulfoxidation of thioanisole were also observed.

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“…Some enzymes can oxidize aliphatic ketones to aliphatic esters, such as the BVMO from Pseudomonas fluorescens DSM 50106 (BVMO Pf ), the BVMO from Pseudomonas putida KT2440 (BVMO Pp ), and the BVMO from Aspergillus flavus NRRL3357 (BVMO AFL838 ) (10)(11)(12). Also, several stable BVMOs have been identified (13,14), and protein engineering has been employed to improve the properties of BVMOs (15), including expanding the substrate scope (16)(17)(18)(19), enhancing regioselectivity (20)(21)(22) or enantioselectivity (23)(24)(25), stimulating sulfoxidation activity (26)(27)(28), changing the cofactor dependency (29)(30)(31), and increasing stability (32)(33)(34).…”

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confidence: 99%

Efficient Synthesis of Methyl 3-Acetoxypropionate by a Newly Identified Baeyer-Villiger Monooxygenase

Liu

et al. 2019

Appl Environ Microbiol

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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 (BVMO Rp ) with a high affinity toward aliphatic methyl ketones (K m Ͻ 3.0 M). 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 (k cat ϭ 2.1 s Ϫ1 ; K m ϭ 1.5 M). Furthermore, BVMO Rp exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMO Rp 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 Ϫ1 h Ϫ1 . Thus, BVMO Rp 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 (BVMO Rp ) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMO Rp 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 BVMO Rp -catalyzed oxidation holds great promise to replace traditional fermentation.

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Hot spots for the protein engineering of Baeyer-Villiger monooxygenases

Cited by 75 publications

References 121 publications

Lactone monomers obtained by enzyme catalysis and their use in reversible thermoresponsive networks

Lactone monomers obtained by enzyme catalysis and their use in reversible thermoresponsive networks

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

Efficient Synthesis of Methyl 3-Acetoxypropionate by a Newly Identified Baeyer-Villiger Monooxygenase

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