Use of isolated cyclohexanone monooxygenase from recombinant <i>Escherichia coli</i> as a biocatalyst for Baeyer–Villiger and sulfide oxidations

Zambianchi, Francesca; Pasta, Piero; Carrea, Giacomo; Colonna, Stefano; Gaggero, Nicoletta; Woodley, John M.

doi:10.1002/bit.10207

Cited by 97 publications

(78 citation statements)

References 27 publications

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“…However, until now it is not certain if the properties of these enzymes allow their in vitro use. Although a few reports concerning the use of oxygenases (for a compilation see [53] ) in synthetic applications do exist, bioengineering-related data are still scarce.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

“…They reached a ttn for NAD + of maximally 503. [53] In cell-free application of cyclohexanone monooxygenase coupled to NADPH regeneration by alcohol dehydrogenase from…”

Section: Discussionmentioning

confidence: 99%

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

et al. 2005

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“…This indicated that the enzyme is in vivo more stable when compared with the isolated enzyme, which might explain the relatively poor stability of many reported isolated coenzymedependent enzymes. For example, a well studied homolog of HAPMO, cyclohexanone monooxygenase, has been shown to inactivate quite rapidly at room temperature (39). The concept of enzyme stabilization by AADP ϩ binding might be exploited for enzyme purification or storage.…”

Section: Discussionmentioning

confidence: 99%

Coenzyme Binding during Catalysis Is Beneficial for the Stability of 4-Hydroxyacetophenone Monooxygenase

Heuvel¹,

Tahallah²,

Kamerbeek

et al. 2005

Journal of Biological Chemistry

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The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP ؉ only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP ؉ interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP ؉ ) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes.The interaction of proteins with small molecules, such as ligands and cofactors, often coincides with an increased stability of the protein due to the coupling of binding with the unfolding equilibrium (1-4). Thus, apart from their catalytic role, cofactors may also have a structural role. The importance of cofactor binding for protein stability is revealed for several flavoproteins (2). The common polymorphism 677TC 3 T in methylenetetrahydrofolate reductase causing the single point mutation A222V reduces the affinity of the enzyme for the FAD cofactor, resulting in a lower thermal stability (5, 6). For the octameric protein vanillylalcohol oxidase, it was demonstrated that cofactor binding influences the quaternary architecture of the enzyme (7). The H61T mutant is purified as apo-enzyme and mainly exists as a dimeric species. The binding of FAD to the enzyme restores the octameric conformation (7,8). Similarly, for lipoamide dehydrogenase, it was shown that FAD binding increases the protein melting temperature from 35 to 80°C (9). Coenzymes such as NAD(P) ϩ and NAD(P)H can have, besides from their function in electron transfer, also a structural role. Several liver and pectoral muscle enzymes are substantially protected by NAD(P) ϩ against heat and proteolysis by trypsin (10). Phosphite dehydrogenase is stabilized by NAD ϩ (11), and DNA ligases become more compact upon anchoring coenzyme NAD ϩ (12).In the present work, we have studied the noncovalent interactions between the oxidized...

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“…[7] One of the main barriers to exploiting CHMO as ab iocatalyst is its lack of stability. [8] To date,t he best-characterized CHMOs are from…”

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confidence: 99%

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

et al. 2016

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Cyclohexanone monooxygenase (CHMO) is ap romising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity.However,the lowstability of many BaeyerVilliger monooxygenases is an obstacle for their exploitation in industry.C haracterization and crystal structure determination of ar obust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts avariety of aliphatic, aromatic,a nd cyclic ketones,a sw ell as prochiral sulfides.A compact substrate-binding cavity explains its preference for small rather than bulky substrates.S mall-scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO.T he exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.

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Use of isolated cyclohexanone monooxygenase from recombinant Escherichia coli as a biocatalyst for Baeyer–Villiger and sulfide oxidations

Cited by 97 publications

References 27 publications

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Coenzyme Binding during Catalysis Is Beneficial for the Stability of 4-Hydroxyacetophenone Monooxygenase

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

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