Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

Kadow, Maria; Balke, Kathleen; Willetts, Andrew; Bornscheuer, Uwe T.; Bäckvall, Jan‐Erling

doi:10.1007/s00253-013-5338-3

Cited by 16 publications

(23 citation statements)

References 56 publications

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“…Type I monooxygenases are FAD and NADPH dependent biocatalysts and belong to the class B flavoenzymes, whereas Type II BVMOs require FMN and NADH for catalysis (Pazmino et al 2010b). Recently, the first crystal structure of a t y p e I I B V M O , n a m e l y 3 , 6 -d i k e t o c a m p h a n e monooxygenase, was published (Isupov et al 2015) which complements the intensive research in this area in the past years (Kadow et al , 2014. Nevertheless, most widely applied in synthetic chemistry are Type I BVMOs and more than 50 protein sequences are available for recombinant expression today.…”

Section: Baeyer-villiger Monooxygenases Classification Of Bvmosmentioning

confidence: 94%

Baeyer-Villiger oxidations: biotechnological approach

Bučko

Gemeiner

Schenkmayerová

et al. 2016

Appl Microbiol Biotechnol

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Baeyer-Villiger monooxygenases (BVMOs) are a very well-known and intensively studied class of flavin-dependent enzymes. Their substrate promiscuity, high chemo-, regio-, and enantioselectivity are prerequisites for the use in synthetic chemistry and should pave the way for successful industrial processes. Nonetheless, only a very limited number of industrial relevant transformations are known, mainly due to the lack of BVMOs stability and cofactor dependency. In this review, we focus on novel BVMO-mediated transformations, BVMOs in cascade type reactions, potential industrial applications, and how limitations have been tackled by the community. Special attention will be put on whole-cell immobilization strategies. We emphasize to bridge recent developments in fundamental research to industrial applications.

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Section: Baeyer-villiger Monooxygenases Classification Of Bvmosmentioning

confidence: 94%

Baeyer-Villiger oxidations: biotechnological approach

Bučko

Gemeiner

Schenkmayerová

et al. 2016

Appl Microbiol Biotechnol

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“…The results show that EziG™ is suitable for immobilization of CalA and further optimizations with subsequent applications are ongoing. The FMNH 2 -dependent BVMO 2,5-diketocamphane monooxygenase from Pseudomonas putida (2,5-DKCMO) 32 was coimmobilized on EziG™ with two cofactor-reconverting enzymes (flavin reductase (FRE) from E. coli 33,34 for FMNH 2 recycling and alanine dehydrogenase (AlaDH) from B. subtilis 35 for NADH recycling). This immobilization was performed by exposing the EziG™ carrier to a mixture of all three cell lysates after overexpression of the three enzymes.…”

Section: Methodsmentioning

confidence: 99%

A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications

et al. 2014

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“…3. Comparative sequence data for some proteins purified from camphor-grown P. putida NCIMB 10007 in this study and relevant proteins purified from either camphor-grown P. putida ATCC 17453 (NCIMB 10007) by Iwaki et al (2013) or E. coli BL21(DE3) by Kadow et al (2014). Identical amino acid residues are underlined and shown in bold; equivalent amino acid residues are italicized.…”

Section: Growth Of P Putida Ncimb 10007 On Various Camphor-based Minmentioning

confidence: 99%

“…Two recent developments have challenged further the long-standing functional relationship of the DKCMOs and the monomeric 36 kDa NADH dehydrogenase first proposed by Gunsalus et al (1965a). Recombinant expression studies by Kadow et al (2011Kadow et al ( , 2014 demonstrated that the genes encoding the biooxygenating subunits of both the 2,5-and 3,6-DKCMO isoenzymes were functional when expressed in E. coli by co-opting Fre, a native FR from the surrogate strain. Of even greater relevance, it has been shown that the biooxygenating subunits of both DKCMO isoenzymes can perform highly efficient lactone-forming biotransformations in association with a camphor-induced homodimeric FR (26~18 kDa) detected in the cells of NCIMB 10007 (ATCC 17435) entering into the stationary phase of growth in strict contrast to the monomeric 36 kDa NADH dehydrogenase reported previously in equivalent cells (vide infra).…”

Section: Introductionmentioning

confidence: 99%

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes

Willetts

Kelly

2014

Microbiology

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Although they have been studied for nearly 50 years, the source of the FMNH 2 needed for effective biooxidation by the 2,5-and 3,6-diketocamphane monooxygenase (DKCMO) isoenzymes induced by the growth of Pseudomonas putida NCIMB 10007 (ATCC 17453) on camphor remains incompletely characterized. Prior studies have focussed exclusively on enzymes present in cells harvested during late-exponential-phase growth despite considerable circumstantial evidence that the flavin reductase (FR) component of these multicomponent monooxygenases is subject to growth-phase-dependent variation. In this study, a number of alternative FMNH 2 -generating activities, including both conventional FRs and enzymes also able to serve as ferric reductases, were isolated from camphor-grown cells, and the relative level, and hence potential contribution, of these various proteins shown to vary considerably depending on the point of harvest of NCIMB 10007 within exponential-phase growth. While two constitutive monomeric ferric reductases (molecular masses 27.0 and 28.5 kDa) were found to be the major relevant sources of FMNH 2 during the initial stages of growth on camphor-based media, a significant subsequent contribution throughout the mid-to late-exponential phases of growth was also made by the camphor-induced homodimeric 37.0 kDa FR Fred, recently reported to serve such a role exclusively. The possible involvement of camphor-induced putidaredoxin reductase (51.0 kDa) as a contributory activity was also investigated and considered. Studies with highly purified preparations of the isofunctional DKCMOs confirmed the potential of the various reductases to function effectively as sources of the requisite FMNH 2 to both monooxygenases at different times throughout growth on camphor.

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Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

Cited by 16 publications

References 56 publications

Baeyer-Villiger oxidations: biotechnological approach

Baeyer-Villiger oxidations: biotechnological approach

A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes

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