Journal articles: 'Type II Baeyer-Villiger monooxygenase'

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Isupov, Michail N., Ewald Schröder, Robert P. Gibson, Jean Beecher, Giuliana Donadio, Vahid Saneei, Stephlina A. Dcunha, et al. "The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid ofPseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase." Acta Crystallographica Section D Biological Crystallography 71, no. 11 (October 31, 2015): 2344–53. http://dx.doi.org/10.1107/s1399004715017939.

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The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid ofPseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in acisconformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

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Isupov, Michail N., Ewald Schröder, Robert P. Gibson, Jean Beecher, Giuliana Donadio, Vahid Saneei, Stephlina A. Dcunha, et al. "The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid ofPseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase. Corrigendum." Acta Crystallographica Section D Structural Biology 74, no. 4 (April 1, 2018): 379. http://dx.doi.org/10.1107/s205979831800150x.

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Löwe, Jana, Olga Blifernez-Klassen, Thomas Baier, Lutz Wobbe, Olaf Kruse, and Harald Gröger. "Type II flavoprotein monooxygenase PsFMO_A from the bacterium Pimelobacter sp. Bb-B catalyzes enantioselective Baeyer-Villiger oxidations with a relaxed cofactor specificity." Journal of Biotechnology 294 (March 2019): 81–87. http://dx.doi.org/10.1016/j.jbiotec.2019.01.011.

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Riebel, Anette, Michael J. Fink, Marko D. Mihovilovic, and Marco W. Fraaije. "Type II Flavin-Containing Monooxygenases: A New Class of Biocatalysts that Harbors Baeyer-Villiger Monooxygenases with a Relaxed Coenzyme Specificity." ChemCatChem 6, no. 4 (October 7, 2013): 1112–17. http://dx.doi.org/10.1002/cctc.201300550.

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Tanner, Adam, and David J. Hopper. "Conversion of 4-Hydroxyacetophenone into 4-Phenyl Acetate by a Flavin Adenine Dinucleotide-Containing Baeyer-Villiger-Type Monooxygenase." Journal of Bacteriology 182, no. 23 (December 1, 2000): 6565–69. http://dx.doi.org/10.1128/jb.182.23.6565-6569.2000.

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ABSTRACT An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M r of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent Km values of 47 μM for 4-hydroxyacetophenone, 384 μM for acetophenone, and 23 μM for 4-hydroxypropiophenone. The apparentKm value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 μM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from anAcinetobacter sp.

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Niero, Mattia, Irene Righetto, Elisa Beneventi, Patrizia Polverino de Laureto, Marco Wilhelmus Fraaije, Francesco Filippini, and Elisabetta Bergantino. "Unique Features of a New Baeyer–Villiger Monooxygenase from a Halophilic Archaeon." Catalysts 10, no. 1 (January 16, 2020): 128. http://dx.doi.org/10.3390/catal10010128.

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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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Iwaki, Hiroaki, Yoshie Hasegawa, Shaozhao Wang, Margaret M. Kayser, and Peter C. K. Lau. "Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli-Expressed Cyclopentanone 1,2-Monooxygenase." Applied and Environmental Microbiology 68, no. 11 (November 2002): 5671–84. http://dx.doi.org/10.1128/aem.68.11.5671-5684.2002.

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ABSTRACT Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus Comamonas. Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as (orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orf3-orf4 [partial 3′ end]). The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ-Kmr cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of σ54-dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-β-thio-d-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.

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Kostichka, Kristy, Stuart M. Thomas, Katharine J. Gibson, Vasantha Nagarajan, and Qiong Cheng. "Cloning and Characterization of a Gene Cluster for Cyclododecanone Oxidation in Rhodococcus ruber SC1." Journal of Bacteriology 183, no. 21 (November 1, 2001): 6478–86. http://dx.doi.org/10.1128/jb.183.21.6478-6486.2001.

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ABSTRACT Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. colistrains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C11 to C15), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C5 to C7).

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Iwaki, Hiroaki, Shaozhao Wang, Stephan Grosse, Hélène Bergeron, Ayako Nagahashi, Jittiwud Lertvorachon, Jianzhong Yang, Yasuo Konishi, Yoshie Hasegawa, and Peter C. K. Lau. "Pseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic Ketones." Applied and Environmental Microbiology 72, no. 4 (April 2006): 2707–20. http://dx.doi.org/10.1128/aem.72.4.2707-2720.2006.

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ABSTRACT Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ∼60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 μmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ∼1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (Km = 8 μM versus Km = 24 μM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C11 to C15 ketones, methyl-substituted C5 and C6 ketones, and bicyclic ketones, such as decalone and β-tetralone. CPDMO has the highest affinity (Km = 5.8 μM) and the highest catalytic efficiency (k cat/Km ratio of 7.2 × 105 M−1 s−1) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.

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Tolmie, Carmien, Martha Smit, and Diederik Opperman. "Alternative Splicing of the Aflatoxin-Associated Baeyer–Villiger Monooxygenase from Aspergillus flavus: Characterisation of MoxY Isoforms." Toxins 10, no. 12 (December 5, 2018): 521. http://dx.doi.org/10.3390/toxins10120521.

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Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type I Baeyer–Villiger monooxygenase (BVMO), responsible for the conversion of hydroxyversicolorone (HVN) and versicolorone (VN) to versiconal hemiacetal acetate (VHA) and versiconol acetate (VOAc), respectively. Using mRNA data, an intron near the C-terminus was identified that is alternatively spliced, creating two possible MoxY isoforms which exist in vivo, while analysis of the genomic DNA suggests an alternative start codon leading to possible elongation of the N-terminus. These four variants of the moxY gene were recombinantly expressed in Escherichia coli, and their activity evaluated with respect to their natural substrates HVN and VN, as well as surrogate ketone substrates. Activity of the enzyme is absolutely dependent on the additional 22 amino acid residues at the N-terminus. Two MoxY isoforms with alternative C-termini, MoxYAltN and MoxYAltNC, converted HVN and VN, in addition to a range of ketone substrates. Stability and flavin-binding data suggest that MoxYAltN is, most likely, the dominant isoform. MoxYAltNC is generated by intron splicing, in contrast to intron retention, which is the most prevalent type of alternative splicing in ascomycetes. The alternative C-termini did not alter the substrate acceptance profile, or regio- or enantioselectivity of the enzyme, but did significantly affect the solubility and stability.

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Willetts, Andrew, Ian Joint, Jack A. Gilbert, William Trimble, and Martin Mühling. "Isolation and initial characterization of a novel type of Baeyer-Villiger monooxygenase activity from a marine microorganism." Microbial Biotechnology 5, no. 4 (March 13, 2012): 549–59. http://dx.doi.org/10.1111/j.1751-7915.2012.00337.x.

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Zhang, Chunyan, Huaran Zhang, and Jianhua Ju. "On-PKS Baeyer–Villiger-Type O-Atom Insertion Catalyzed by Luciferase-Like Monooxygenase OvmO during Olimycin Biosynthesis." Organic Letters 22, no. 5 (February 19, 2020): 1780–84. http://dx.doi.org/10.1021/acs.orglett.0c00076.

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Dudek, Hanna M., Gonzalo de Gonzalo, Daniel E. Torres Pazmiño, Piotr Stępniak, Lucjan S. Wyrwicz, Leszek Rychlewski, and Marco W. Fraaije. "Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis." Applied and Environmental Microbiology 77, no. 16 (July 1, 2011): 5730–38. http://dx.doi.org/10.1128/aem.00687-11.

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ABSTRACTBaeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) fromThermobifida fuscais the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

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Leisch, Hannes, Rong Shi, Stephan Grosse, Krista Morley, Hélène Bergeron, Miroslaw Cygler, Hiroaki Iwaki, Yoshie Hasegawa, and Peter C. K. Lau. "Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453." Applied and Environmental Microbiology 78, no. 7 (January 20, 2012): 2200–2212. http://dx.doi.org/10.1128/aem.07694-11.

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ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

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Mansouri, Hamid R., Marko D. Mihovilovic, and Florian Rudroff. "Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability." ChemBioChem 21, no. 7 (April 2020): 971–77. http://dx.doi.org/10.1002/cbic.201900501.

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Weiss, Michael, Karin Denger, Thomas Huhn, and David Schleheck. "Two Enzymes of a Complete Degradation Pathway for Linear Alkylbenzenesulfonate (LAS) Surfactants: 4-Sulfoacetophenone Baeyer-Villiger Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1." Applied and Environmental Microbiology 78, no. 23 (September 21, 2012): 8254–63. http://dx.doi.org/10.1128/aem.02412-12.

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ABSTRACTComplete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C4-SPC). Second, these SPCs are mineralized. 3-C4-SPC is mineralized byComamonas testosteroniKF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C4-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria.

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Landry, Zachary, Brandon K. Swan, Gerhard J. Herndl, Ramunas Stepanauskas, and Stephen J. Giovannoni. "SAR202 Genomes from the Dark Ocean Predict Pathways for the Oxidation of Recalcitrant Dissolved Organic Matter." mBio 8, no. 2 (April 18, 2017). http://dx.doi.org/10.1128/mbio.00413-17.

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ABSTRACTDeep-ocean regions beyond the reach of sunlight contain an estimated 615 Pg of dissolved organic matter (DOM), much of which persists for thousands of years. It is thought that bacteria oxidize DOM until it is too dilute or refractory to support microbial activity. We analyzed five single-amplified genomes (SAGs) from the abundant SAR202 clade of dark-ocean bacterioplankton and found they encode multiple families of paralogous enzymes involved in carbon catabolism, including several families of oxidative enzymes that we hypothesize participate in the degradation of cyclic alkanes. The five partial genomes encoded 152 flavin mononucleotide/F420-dependent monooxygenases (FMNOs), many of which are predicted to be type II Baeyer-Villiger monooxygenases (BVMOs) that catalyze oxygen insertion into semilabile alicyclic alkanes. The large number of oxidative enzymes, as well as other families of enzymes that appear to play complementary roles in catabolic pathways, suggests that SAR202 might catalyze final steps in the biological oxidation of relatively recalcitrant organic compounds to refractory compounds that persist.IMPORTANCECarbon in the ocean is massively sequestered in a complex mixture of biologically refractory molecules that accumulate as the chemical end member of biological oxidation and diagenetic change. However, few details are known about the biochemical machinery of carbon sequestration in the deep ocean. Reconstruction of the metabolism of a deep-ocean microbial clade, SAR202, led to postulation of new biochemical pathways that may be the penultimate stages of DOM oxidation to refractory forms that persist. These pathways are tied to a proliferation of oxidative enzymes. This research illuminates dark-ocean biochemistry that is broadly consequential for reconstructing the global carbon cycle.

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Ceccoli, Romina D., Dario A. Bianchi, Michael J. Fink, Marko D. Mihovilovic, and Daniela V. Rial. "Cloning and characterization of the Type I Baeyer–Villiger monooxygenase from Leptospira biflexa." AMB Express 7, no. 1 (April 27, 2017). http://dx.doi.org/10.1186/s13568-017-0390-5.

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Zhang, Yan, Feng Liu, Na Xu, Yin-Qi Wu, Yu-Cong Zheng, Qian Zhao, Guoqiang Lin, Hui-Lei Yu, and Jian-He Xu. "Discovery of Two Native Baeyer-Villiger Monooxygenases for Asymmetric Synthesis of Bulky Chiral Sulfoxides." Applied and Environmental Microbiology 84, no. 14 (May 11, 2018). http://dx.doi.org/10.1128/aem.00638-18.

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ABSTRACT Two Baeyer-Villiger monooxygenases (BVMOs), designated Bo BVMO and Am BVMO, were discovered from Bradyrhizobium oligotrophicum and Aeromicrobium marinum , respectively. Both monooxygenases displayed novel features for catalyzing the asymmetric sulfoxidation of bulky and pharmaceutically relevant thioethers. Evolutionary relationship and sequence analysis revealed that the two BVMOs belong to the family of typical type I BVMOs and the subtype ethionamide monooxygenase. Both BVMOs are active toward medium- and long-chain aliphatic ketones as well as various thioether substrates but are ineffective toward cyclohexanone, aromatic ketones, and other typical BVMO substrates. Bo BVMO and Am BVMO showed the highest activities (0.117 and 0.025 U/mg protein, respectively) toward thioanisole among the tested substrates. Furthermore, these BVMOs exhibited distinct activity and excellent stereoselectivity toward bulky and prochiral prazole thioethers, which is a unique feature of this family of BVMOs. No native enzyme has been reported for the asymmetric sulfoxidation of bulky prazole thioethers into chiral sulfoxides. The identification of Bo BVMO and Am BVMO provides an important scaffold for discovering enzymes capable of asymmetrically oxidizing bulky thioether substrates by genome mining. IMPORTANCE Baeyer-Villiger monooxygenases (BVMOs) are valuable enzyme catalysts that are an alternative to the chemical Baeyer-Villiger oxidation reaction. Although BVMOs display broad substrate ranges, no native enzymes were reported to have activity toward the asymmetric oxidation of bulky prazole-like thioether substrates. Herein, we report the discovery of two type I BVMOs from Bradyrhizobium oligotrophicum ( Bo BVMO) and Aeromicrobium marinum ( Am BVMO) which are able to catalyze the asymmetric sulfoxidation of bulky prazole thioethers (proton pump inhibitors [PPIs], a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production). Efficient catalysis of omeprazole oxidation by Bo BVMO was developed, indicating that this enzyme is a promising biocatalyst for the synthesis of bulky and pharmaceutically relevant chiral sulfoxide drugs. These results demonstrate that the newly identified enzymes are suitable templates for the discovery of more and better thioether-converting BVMOs.

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Qin, Peng, Xin‐Yi Lu, Jian‐He Xu, and Hui‐Lei Yu. "Directed evolution of Baeyer–Villiger monooxygenase for highly secretory expressed in Pichia pastoris and efficient preparation of chiral pyrazole sulfoxide." Biotechnology and Bioengineering, December 13, 2023. http://dx.doi.org/10.1002/bit.28617.

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AbstractThe methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high‐density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer–Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5‐fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence‐activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C‐terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild‐type. Finally, the optimized chassis cells were used for high‐density fermentation on a 5‐L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH‐adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)‐sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.

Journal articles on the topic 'Type II Baeyer-Villiger monooxygenase'

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