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Among the molecular mechanisms of adaptation in biology, enzyme functional diversification is indispensable. By allowing organisms to expand their catalytic repertoires and adopt fundamentally different chemistries, animals can harness or eliminate new-found substances and xenobiotics that they are exposed to in new environments. Here, we explore the flavin-containing monooxygenases (FMOs) that are essential for xenobiotic detoxification. Employing a paleobiochemistry approach in combination with enzymology techniques we disclose the set of historical substitutions responsible for the family’s functional diversification in tetrapods. Remarkably, a few amino acid replacements differentiate an ancestral multi-tasking FMO into a more specialized monooxygenase by modulating the oxygenating flavin intermediate. Our findings substantiate an ongoing premise that enzymatic function hinges on a subset of residues that is not limited to the active site core.

The asymmetric hydrogenation of conjugated tetrasubstituted alkenes with transition-metal catalysts is a challenging reaction, especially for substrates bearing a halide substituent. We describe a two-step multienzymatic reduction of a series of α-halo β-alkyl tetrasubstituted cyclic enones, affording halohydrins with three contiguous stereogenic centers, in good yield and with a high stereoselectivity. The reduction is catalyzed by a stereospecific ene-reductase (OYE2-3 or NemA) and a highly enantioselective alcohol dehydrogenase (ADH). The use of two enantiodivergent ADHs allows the control of the diastereoselectivity. The absolute stereochemical configurations of the products have been determined from the analysis of single-crystal structures (Flack's parameter). The enantiomeric excess (ee) has been determined by derivatization of the products with (R) Mosher's acid. Lastly, we extended our methodology also to a nonhalogenated substrate: the α-methyl ketoisophorone was reduced by two distinct enantiodivergent ene-reductases (flavin mononucleotide- and F 420-dependent), affording each enantiomer of the saturated ketone with ee > 98%.

Flavoprotein oxidases can catalyze oxidations of alcohols and amines by merely using molecular oxygen as the oxidant, making this class of enzymes appealing for biocatalysis. The FAD‐containing (FAD=flavin adenine dinucleotide) alcohol oxidase from P. chrysosporium facilitated double and triple oxidations for a range of aliphatic diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. For example, diethylene glycol could be selectively and fully converted into 2‐(2‐hydroxyethoxy)acetic acid. Such a facile cofactor‐independent biocatalytic route towards hydroxy acids opens up new avenues for the preparation of polyester building blocks., Triple action: The flavoprotein alcohol oxidase (AOX*) facilitates the double and triple oxidations of diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. Such biocatalytic route towards hydroxy acids has potential for the preparation of polyester building blocks.

The azoreductase AzoA from the alkali-tolerant Bacillus wakoensis A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates (kcat values of 100-400 s-1 for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases.

Discovery of novel enzymes is a challenging task, yet a crucial one, due to their increasing relevance as chemical catalysts and biotechnological tools. In our work we present a high-throughput screening approach to discovering novel activities. A screen of 96 putative oxidases with 23 substrates led to the discovery of two new enzymes. The first enzyme, N-acetyl-D-hexosamine oxidase (EC 1.1.3.29) from Ralstonia solanacearum, is a vanillyl alcohol oxidase-like flavoprotein displaying the highest activity with N-acetylglucosamine and N-acetylgalactosamine. Before our discovery of the enzyme, its activity was an orphan one - experimentally characterized but lacking the link to amino acid sequence. The second enzyme, from an uncultured marine euryarchaeota, is a long-chain alcohol oxidase (LCAO, EC 1.1.3.20) active with a range of fatty alcohols, with 1-dodecanol being the preferred substrate. The enzyme displays no sequence similarity to previously characterised LCAOs, and thus is a completely novel representative of a protein with such activity.

L-Amino acid oxidase (LAAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme active on most proteinogenic L-amino acids, catalysing their conversion to α-keto acids by oxidative deamination of the substrate. For this oxidation reaction, molecular oxygen is used as the electron acceptor, generating hydrogen peroxide. LAAO can be used to detect L-amino acids, for the production of hydrogen peroxide as an oxidative agent or antimicrobial agent, and for the production of enantiopure amino acids from racemates. In this work, we characterised a previously reported LAAO from the bacterium Pseudoalteromonas luteoviolacea. The substrate scope and kinetic properties of the enzyme were determined, and the thermostability was evaluated. Additionally, we elucidated the crystal structure of this bacterial LAAO, enabling us to test the role of active site residues concerning their function in catalysis. The obtained insights and ease of expression of this thermostable LAAO provides a solid basis for the development of engineered LAAO variants tuned for biosensing and/or biocatalysis.

Funding Information: Acknowledgments: We acknowledge the support from Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE 2020-Programa Operacional Competitividade e Internacionalização (POCI), from FCT—Fundação para a Ciência e a Tecnologia (PTDC/BIA-BFS/31026/2017 and 2020.05017.BD) and from the European Union’s Horizon 2020 Research and Innovation Program, through TIMB3 and B-LigZymes projects (grant agreements numbers are 810856 and 824017, respectively). Molecular graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Funding Information: This research was funded by FCT?Funda??o para a Ci?ncia e a Tecnologia (grant number: PTDC/BIA-BFS/31026/2017). The APC was funded by the TIMB3 project, European Union?s Horizon 2020 Research and Innovation Program (grant agreement No.: 810856). Funding Information: Funding: This research was funded by FCT—Fundação para a Ciência e a Tecnologia (grant number: PTDC/BIA-BFS/31026/2017). The APC was funded by the TIMB3 project, European Union’s Horizon 2020 Research and Innovation Program (grant agreement No.: 810856). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2 . To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2 . While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the im-mobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates. publishersversion published

The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD–RIAD) system. These peptides allow self-assembly based on specific protein–protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer–Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in E. coli and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMORIAD–PTDHRIDD and PAMORIAD/RIAD–PTDHRIDD). These enzyme complexes displayed similar catalytic activity when compared with the PTDH–PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO–PTDH complex less PTDH was required for the same performance when compared with the PTDH–PAMO fusion enzyme.

Pollution, accidents, and misinformation have earned the pharmaceutical and chemical industry a poor public reputation, despite their undisputable importance to society. Biotechnological advances hold the promise to enable a future of drastically reduced environmental impact and rigorously more efficient production routes at the same time. This is exemplified in the Baeyer-Villiger reaction, which offers a simple synthetic route to oxidize ketones to esters, but application is hampered by the requirement of hazardous and dangerous reagents. As an attractive alternative, flavin-containing Baeyer-Villiger monooxygenases (BVMOs) have been investigated for their potential as biocatalysts for a long time, and many variants have been characterized. After a general look at the state of biotechnology, we here summarize the literature on biochemical characterizations, mechanistic and structural investigations, as well as enzyme engineering efforts in BVMOs. With a focus on recent developments, we critically outline the advances toward tuning these enzymes suitable for industrial applications.

Enzymes often by far exceed the activity, selectivity, and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature‐sensitive enzymes catalyzing the Baeyer–Villiger reaction, which display excellent stereo‐ and regioselectivity and offer a green alternative to the commonly used, explosive peracids. Here we describe a protein engineering approach applied to cyclohexanone monooxygenase from Rhodococcus sp. HI‐31, a substrate‐promiscuous enzyme that efficiently catalyzes the production of the nylon‐6 precursor ε‐caprolactone. We used a framework for rapid enzyme stabilization by computational libraries (FRESCO), which predicts protein‐stabilizing mutations. From 128 screened point mutants, approximately half had a stabilizing effect, albeit mostly to a small degree. To overcome incompatibility effects observed upon combining the best hits, an easy shuffled library design strategy was devised. The most stable and highly active mutant displayed an increase in unfolding temperature of 13°C and an approximately 33x increase in half‐life at 30°C. In contrast to the wild‐type enzyme, this thermostable 8x mutant is an attractive biocatalyst for biotechnological applications., This work describes the stabilization of an e‐caprolactone‐producing cyclohexanone monooxygenase through protein engineering. Applying a computational pipeline for predicting stabilizing point mutations and an innovative shuffled library strategy for their combination, the authors report an increase of 13 °C of the melting temperature of the notoriously unstable enzyme, while the half‐life improved 33‐fold.

F-420-dependent enzymes are found in many microorganisms and can catalyze a wide range of redox reactions, including those with some substrates that are otherwise recalcitrant to enzyme-mediated reductions. Unfortunately, the scarceness of the cofactor prevents application of these enzymes in biocatalysis. The best F-420-producing organism, Mycobacterium smegmatis, only produces 1.4 mu mol per liter of culture. Therefore, we synthesized the unnatural cofactor FO-5'-phosphate, coined FOP. The FO core-structure was chemically synthesized, and an engineered riboflavin kinase from Corynebacterium ammoniagenes (CaRFK) was then used to phosphorylate the 5'-hydroxyl group. The triple F21H/F85H/A66I CaRFK mutant reached 80% of FO conversion in 12 h. The same enzyme could produce 1 mg (2.5 mu mol) of FOP in 50 mL of reaction volume, which translates to a production of 50 mu mol/L. The activity toward FOP was tested for an enzyme of each of the three main structural classes of F-420-dependent oxidoreductases. The sugar-6-phosphate dehydrogenase from Cryptosporangium arvum (FSD-Cryar), the F-420:NADPH oxidoreductase from Thermobifida fusca (TfuFNO), and the F-420-dependent reductases from Mycobacterium hassiacum (FDR-Mha) all showed activity for FOP. Although the activity for FOP was lower than that for F-420, with slightly lower k(cat) and higher K-m values, the catalytic efficiencies were only 2.0, 12.6, and 22.4 times lower for TfuFNO, FSD-Cryar, and FDR-Mha, respectively. Thus, FOP could be a serious alternative for replacing F-420 and might boost the application of F-420-dependent enzymes in biocatalysis.

Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated form NADP are crucial cofactors for a large array of biocatalytically important redox enzymes. Their high cost and relatively poor stability, however, make them less attractive electron mediators for industrial processes. Nicotinamide cofactor biomimetics (NCBs) are easily synthesized, are inexpensive, and are also generally more stable than their natural counterparts. A bottleneck for the application of these artificial hydride carriers is the lack of efficient cofactor recycling methods. Therefore, we engineered the thermostable F420:NADPH oxidoreductase from Thermobifida fusca (Tfu-FNO), by structure-inspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor F420H2 was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with Km values in the millimolar range and kcat values ranging from 0.09 to 1.4 min-1. Saturation mutagenesis at positions Gly-29 and Pro-89 resulted in mutants with up to 139 times higher catalytic efficiencies. Mutant G29W showed a kcat value of 4.2 s-1 toward 1-benzyl-3-acetylpyridine (BAP+), which is similar to the kcat value for the natural substrate NADP+. The best Tfu-FNO variants for a specific NCB were then used for the recycling of catalytic amounts of these nicotinamides in conversion experiments with the thermostable ene-reductase from Thermus scotoductus (TsOYE). We were able to fully convert 10 mM ketoisophorone with BAP+ within 16 h, using F420 or its artificial biomimetic FOP (FO-2′-phosphate) as an efficient electron mediator and glucose-6-phosphate as an electron donor. The generated toolbox of thermostable and NCB-dependent Tfu-FNO variants offers powerful cofactor regeneration biocatalysts for the reduction of several artificial nicotinamide biomimetics at both ambient and high temperatures. In fact, to our knowledge, this enzymatic method seems to be the best-performing NCB-recycling system for BNAH and BAPH thus far.

The enantioselective oxidation of secondary alcohols represents a valuable approach for the synthesis of optically pure compounds. Flavoprotein oxidases can catalyse such selective transformations by merely using oxygen as electron acceptor. While many flavoprotein oxidases preferably act on primary alcohols, the FAD-containing alcohol oxidase from Phanerochaete chrysosporium was found to be able to perform kinetic resolutions of several secondary alcohols. By selective oxidation of the (S)-alcohols, the (R)-alcohols were obtained in high enantiopurity. In silico docking studies were carried out in order to substantiate the observed (S)-selectivity. Several hydrophobic and aromatic residues in the substrate binding site create a cavity in which the substrates can comfortably undergo van der Waals and pi-stacking interactions. Consequently, oxidation of the secondary alcohols is restricted to one of the two enantiomers. This study has uncovered the ability of an FAD-containing alcohol oxidase, that is known for oxidizing small primary alcohols, to perform enantioselective oxidations of various secondary alcohols.

This chapter represents a journey through flavoprotein oxidases. The purpose is to excite the reader curiosity regarding this class of enzymes by showing their diverse applications. We start with a brief overview on oxidases to then introduce flavoprotein oxidases and elaborate on the flavin cofactors, their redox and spectroscopic characteristics, and their role in the catalytic mechanism. The six major flavoprotein oxidase families will be described, giving examples of their importance in biology and their biotechnological uses. Specific attention will be given to a few selected flavoprotein oxidases that are not extensively discussed in other chapters of this book. Glucose oxidase, cholesterol oxidase, 5-(hydroxymethyl)furfural (HMF) oxidase and methanol oxidase are four examples of oxidases belonging to the GMC-like flavoprotein oxidase family and that have been shown to be valuable biocatalysts. Their structural and mechanistic features and recent enzyme engineering will be discussed in details. Finally we give a look at the current trend in research and conclude with a future outlook.

Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer–Villiger‐type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans‐AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans‐AT PKSs in the specialized metabolism of symbiotic bacteria., O where art thou: In the classic model for modular polyketide synthases (PKSs), a small set of reactions generate combinatorially substituted carbon chains. Architecturally diverse PKS modules were identified that diversify polyketides by oxygenation. An orphan PKS with two oxygenase modules from a plant symbiont was found to produce lobatamides, which are in‐chain‐oxygenated oxime‐bearing cytotoxins that were previously only known from a tunicate.

Type I Baeyer&ndash, 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 silicoanalyses, 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&ndash, 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.

Deep eutectic solvents (DESs) have been applied as cosolvents in various biocatalytic processes during recent years. However, their use in combination with redox enzymes has been limited. In this study, we have explored the beneficial effects of several DES as cosolvents on the performance of 5-hydroxymethylfurfural oxidase (HMFO), a valuable oxidative enzyme for the preparation of furan-2,5-dicarboxylic acid (FDCA), and other compounds, such as carbonyl compounds and carboxylic acids. The use of natural DESs, based on glucose and fructose, was found to have a positive effect. Higher conversions are obtained for the synthesis of several oxidized compounds, including FDCA. Depending on the type of DES, the stability of HMFO could be significantly improved. As the use of DES increases the solubility of many substrates while they only mildly affect dioxygen solubility, this study demonstrates that biocatalysis based on HMFO and other redox biocatalysts can benefit from a carefully selected DES.

Glycerol is a major byproduct of biodiesel production, and enzymes that oxidize this compound have been long sought after. The recently described alcohol oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here, we describe the comprehensive structural and biochemical characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active toward methanol, ethanol, and 1-propanol (kcat = 18, 19, and 11 s–1, respectively), but showed very limited activity toward glycerol (kobs = 0.2 s–1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was determined at a resolution of 2.6 Å. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the observed preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity toward glycerol. Among them, the F101S variant had a high kcat value of 3 s–1, retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation.

Indigo is currently produced by a century-old petrochemical-based process, therefore it is highly attractive to develop a more environmentally benign and efficient biotechnological process to produce this timeless dye. Flavin-containing monooxygenases (FMOs) are able to oxidize a wide variety of substrates. In this paper we show that the bacterial mFMO can be adapted to improve its ability to convert indole into indigo. The improvement was achieved by a combination of computational and structure-inspired enzyme redesign. We showed that the thermostability and the kcat for indole could be improved 1.5-fold by screening a relatively small number of enzyme mutants. This project not only resulted in an improved biocatalyst but also provided an improved understanding of the structural elements that determine the activity of mFMO and provides hints for further improvement of the monooxygenase as biocatalyst.

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec-thiols by using structure-guided engineering of 5-(hydroxymethyl) furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec-thiols, thus yielding the corresponding thioketones and nonreacted R-configured thiols with excellent enantioselectivities (E >= 200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen-bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec-thiols.

In the past decade it has become clear that many microbes harbor enzymes that employ an unusual flavin cofactor, the F-420 deazaflavin cofactor. Herein we show that F-420-dependent reductases (FDRs) can successfully perform enantio-, regio- and chemoselective ene-reductions. For the first time, we have demonstrated that F420H2-driven reductases can be used as biocatalysts for the reduction of ,-unsaturated ketones and aldehydes with good conversions (>99%) and excellent regioselectivities and enantiomeric excesses (>99% ee). Noteworthily, FDRs typically display an opposite enantioselectivity when compared to the well established FMN-dependent Old Yellow Enzymes (OYEs).

recently discovered, moderately thermostable Baeyer-Villiger monooxygenase, polycyclic ketone monooxygenase (PockeMO), from Thermothelomyces thermophila has been employed as a biocatalyst in a set of asymmetric sulfoxidations. The enzyme was able to catalyze the oxidation of various alkyl aryl sulfides with good selectivities and moderate to high activities. The biocatalytic performance was able to be further increased by optimizing some reaction parameters, such as the addition of 10% v v− 1 of water miscible solvents or toluene, or by performing the conversion at a relatively high temperature (45 °C). PockeMO was found to display an optimum activity at sulfide concentrations of 50 mM, while it can also function at 200 mM. Taken together, the data show that PockeMO can be used as robust biocatalyst for the synthesis of optically active sulfoxides. European Union 635734 European Union H2020-LEIT BIO-2014-1

Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these challenges, but known BVMOs have shortcomings in either stability or substrate selectivity. We characterized a novel BVMO from the thermophilic fungus Thermothelomyces thermophila, determined its three-dimensional structure, and demonstrated its use as a promising biocatalyst. This fungal enzyme displays excellent enantioselectivity, acts on various ketones, and is particularly active on polycyclic molecules. Most notably we observed that the enzyme can perform oxidations on both the A and D ring when converting steroids. These functional properties can be linked to unique structural features, which identify enzymes acting on bulky substrates as a distinct subgroup of the BVMO class.

Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio‐, chemo‐, and enantioselectivity. However, the low stability of many Baeyer–Villiger monooxygenases is an obstacle for their exploitation in industry. Characterization and crystal structure determination of a robust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts a variety of aliphatic, aromatic, and cyclic ketones, as well as prochiral sulfides. A compact substrate‐binding cavity explains its preference for small rather than bulky substrates. Small‐scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO. The exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.

Highlights•Dye decolorizing peroxidase TfuDyP binds heme and protoporphyrin IX in vivo.•The activity of TfuDyP is dependent on the expression level in E. coli.•Expression of fully functional DyPs can be tuned by the type of expression host and expression conditions.The heterologous overexpression level of the bacterial dye decolorizing peroxidase TfuDyP in Escherichia coli was increased sixty fold to approximately 200 mg of purified enzyme per liter culture broth by fusing the enzyme to the small ubiquitin-related modifier protein (SUMO). The highly over expressed SUMO-TfuDyP was, however, almost inactive. Analysis of the enzyme by UV-vis absorption spectroscopy and high-resolution mass spectrometry showed that a large fraction of the highly over-expressed enzyme contained the iron deficient heme precursor protoporphyrin IX (PPIX) instead of heme. Here we show that the activity of the enzyme was dependent on the expression level of the protein. (C) 2016 Elsevier B.V. All rights reserved.

The consumption of dyes is increasing worldwide in line with the increase of population and demand for clothes and other colored products. However, the efficiency of dyeing processes is still poor and results in large amounts of colored effluents. It is desired to develop a portfolio of enzymes which can be used for the treatment of colored wastewaters. Herein, we used genome sequence information to discover a dye-decolorizing peroxidase (DyP) from Pseudomonas fluorescens Pf-01. Two genes putatively encoding for DyPs were identified in the respective genome and cloned for expression in Escherichia coli, of which one (PfDyP B2) could be overexpressed as a soluble protein. PfDyP B2 shows some typical features known for DyPs which includes the ability to convert dyes at the expense of hydrogen peroxide. Interestingly, t-butyl hydroperoxide could be used as an alternative substrate to hydrogen peroxide. Immobilization of PfDyP B2 in calcium-alginate beads resulted in a significant increase in stability: PfDyP B2 retains 80% of its initial activity after 2 h incubation at 50 &deg, C, while the soluble enzyme is inactivated within minutes. PfDyP B2 was also tested with aniline and ethyl diazoacetate as substrates. Based on GC-MS analyses, 30% conversion of the starting material was achieved after 65 h at 30 &deg, C. Importantly, this is the first report of a DyP-catalyzed insertion of a carbene into an N-H bond.

Detoxifying enzymes such as flavin-containing monooxygenases deal with a huge array of highly diverse xenobiotics and toxic compounds. In addition to being of high physiological relevance, these drug-metabolizing enzymes are useful catalysts for synthetic chemistry. Despite the wealth of studies, the molecular basis of their relaxed substrate selectivity remains an open question. Here, we addressed this issue by applying a cumulative alanine mutagenesis approach to cyclohexanone monooxygenase from Thermocrispum municipale, a flavin-dependent Baeyer–Villiger monooxygenase which we chose as a model system because of its pronounced thermostability and substrate promiscuity. Simultaneous removal of up to eight noncatalytic active-site side chains including four phenylalanines had no effect on protein folding, thermostability, and cofactor loading. We observed a linear decrease in activity, rather than a selectivity switch, and attributed this to a less efficient catalytic environment in the enlarged active-site space. Time-resolved kinetic studies confirmed this interpretation. We also determined the crystal structure of the enzyme in complex with a mimic of the reaction intermediate that shows an unaltered overall protein conformation. These findings led us to propose that this cyclohexanone monooxygenase may lack a distinct substrate selection mechanism altogether. We speculate that the main or exclusive function of the protein shell in promiscuous enzymes might be the stabilization and accessibility of their very reactive catalytic intermediates.

In the past decade it has become clear that many microbes harbor enzymes that employ an unusual flavin cofactor, the F420 deazaflavin cofactor. Herein we show that F420-dependent reductases (FDRs) can successfully perform enantio-, regio- and chemoselective ene-reductions. For the first time, we have demonstrated that F420H2-driven reductases can be used as biocatalysts for the reduction of α,β-unsaturated ketones and aldehydes with good conversions (99%) and excellent regioselectivities and enantiomeric excesses (99% ee). Noteworthily, FDRs typically display an opposite enantioselectivity when compared to the well established FMN-dependent Old Yellow Enzymes (OYEs).

Syringaresinol was synthesized in a one-pot conversion containing eugenol oxidase (EUGO) and horseradish peroxidase (HRP) using the relatively cheap 2,6-dimethoxy-4-allylphenol as a substrate. This conversion is fully coupled as the hydrogen peroxide generated from the reaction of EUGO with the substrate is utilized by the HRP to convert the formed sinapyl alcohol into syringaresinol. To improve the performance of EUGO on 2,6-dimethoxy-4-allylphenol, structure-inspired enzyme engineering was performed. This yielded the I427A EUGO mutant that is significantly more efficient with 2,6-dimethoxy-4-allylphenol. The I427A EUGO mutant together with HRP were capable of efficiently producing syringaresinol as a major product. After optimization and upscaling the conversion to a semipreparative scale (1 gr), syringaresinol was obtained in 81% yield.

Controlling the regioselectivity of Baeyer− Villiger (BV) reactions remains an ongoing issue in organic chemistry, be it by synthetic catalysts or enzymes of the type Baeyer−Villiger monooxygenases (BVMOs). Herein, we address the challenging problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as substrates, which are not structurally biased toward abnormal reactivity. Upon applying iterative saturation mutagenesis at sites lining the binding pocket of the thermostable BVMO from Thermocrispum municipale DSM 44069 (TmCHMO) and using 4-phenyl-2-butanone as substrate, the regioselectivity was reversed from 99:1 (wild-type enzyme in favor of the normal product undergoing 2-phenylethyl migration) to 2:98 in favor of methyl migration when applying the best mutant. This also stands in stark contrast to the respective reaction using the synthetic reagent m-CPBA, which provides solely the normal product. Reversal of regioselectivity was also achieved in the BV reaction of two other linear ketones. Kinetic parameters and melting temperatures revealed that most of the evolved mutants retained catalytic activity, as well as thermostability. In order to shed light on the origin of switched regioselectivity in reactions of 4-phenyl-2-butanone and phenylacetone, extensive QM/MM and MD simulations were performed. It was found that the mutations introduced by directed evolution induce crucial changes in the conformation of the respective Criegee intermediates and transition states in the binding pocket of the enzyme. In mutants that destabilize the normally preferred migration transition state, a reversal of regioselectivity is observed. This conformational control of regioselectivity overrides electronic control, which normally causes preferential migration of the group that is best able to stabilize positive charge. The results can be expected to aid future protein engineering of BVMOs.

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O2-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to “over-oxidation” of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis–Menten kinetics were performed to corroborate these findings.

5-Hydroxymethylfurfural oxidase (HMFO) is a flavin-dependent enzyme that catalyzes the oxidation of many aldehydes, primary alcohols, and thiols. The three-step conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid is relevant for the industrial production of biobased polymers. The remarkable wide substrate scope of HMFO contrasts with the enzyme’s precision in positioning the substrate to perform catalysis. We have solved the crystal structure of HMFO at 1.6 Å resolution, which guided mutagenesis experiments to probe the role of the active-site residues in catalysis. Mutations targeting two active-site residues generated engineered forms of HMFO with promising catalytic features, namely enantioselective activities on secondary alcohols and improved 2,5-furandicarboxylic acid yields.

By a targeted enzyme engineering approach, we were able to create an efficient NADPH oxidase from a monooxygenase. Intriguingly, replacement of only one specific single amino acid was sufficient for such a monooxygenase-to-oxidase switch-a complete transition in enzyme activity. Pre-steady-state kinetic analysis and elucidation of the crystal structure of the C65D PAMO mutant revealed that the mutation introduces small changes near the flavin cofactor, resulting in a rapid decay of the peroxyflavin intermediate. The engineered biocatalyst was shown to be a thermostable, solvent tolerant, and effective cofactor-regenerating biocatalyst. Therefore, it represents a valuable new biocatalytic tool.

Flavoprotein oxidases are a diverse class of biocatalysts, most of which catalyze the oxidation of C[BOND]O, C[BOND]N, or C[BOND]C bonds. Flavoprotein oxidases that are known to catalyze the oxidation of C[BOND]S bonds are rare, being limited to enzymes that catalyze the oxidative cleavage of thioethers. Herein, we report that various flavoprotein oxidases, previously thought to solely act on alcohols, also catalyze the oxidation of thiols to thiocarbonyls. These results highlight the versatility of enzymatic catalysis and provide a potential biocatalytic route to reactive thiocarbonyl compounds, which have a variety of applications in synthetic organic chemistry.

A nicotinamide adenine dinucleotide (NADH)-dependent redox-neutral convergent cascade composed of a recently discovered type II flavin-containing monooxygenase (FMO−E) and horse liver alcohol dehydrogenase (HLADH) has been established. Two model reaction cascades were analyzed for the synthesis of γ-butyrolactone and chiral bicyclic lactones. In the former cascade, all substrates were converted into one single product γ-butyrolactone with high atom efficiency. More than 130 mM γ-butyrolactone were obtained when applying 100 mM cyclobutanone and 50 mM 1,4-butanediol in this cascade. In the second cascade where bicyclo[4.2.0]octan-7-one and cis-1,2-cyclohexanedimethanol were coupled, the ketone substrate was converted to the corresponding normal lactone with an ee value of 89–74% (3aS, 7aS) by FMO−E alone and the abnormal lactone with an ee value of >99% (3aR, 7aS) was formed by both HLADH and FMO−E. (Figure presented.).

Furan-2,5-dicarboxylic acid (FDCA) is a biobased platform chemical for the production of polymers. In the past few years, numerous multistep chemical routes have been reported on the synthesis of FDCA by oxidation of 5-hydroxymethylfurfural (HMF). Recently we identified an FAD-dependent enzyme which is active towards HMF and related compounds. This oxidase has the remarkable capability of oxidizing [5-(hydroxymethyl) furan-2-yl] methanol to FDCA, a reaction involving four consecutive oxidations. The oxidase can produce FDCA from HMF with high yield at ambient temperature and pressure. Examination of the underlying mechanism shows that the oxidase acts on alcohol groups only and depends on the hydration of aldehydes for the oxidation reaction required to form FDCA.

International audience; Enzyme-filled polystyrene-b-poly(3-(isocyano-L-alanyl-aminoethyl)thiophene) (PS-b-PIAT) nanoreactors are encapsulated together with free enzymes and substrates in a larger polybutadiene-b-poly(ethylene oxide) (PB-b-PEO) polymersome, forming a multicompartmentalized structure, which shows structural resemblance to the cell and its organelles. An original cofactor-dependent three-enzyme cascade reaction is performed, using either compatible or incompatible enzymes, which takes place across multiple compartments.

Baeyer-Villiger monooxygenases are attractive "green" catalysts able to produce chiral esters or lactones starting from ketones. They can act as natural equivalents of peroxyacids that are the catalysts classically used in the organic synthesis reactions, consisting in the cleavage of C-C bonds with the concomitant insertion of an oxygen atom.In this study, two type I BVMOs have been identified for the first time in photosynthetic eukaryotic organisms, the red alga Cyanidioschyzon merolae (Cm) and the moss Physcomitrella patens (Pp). A biocatalytic characterization of these newly discovered enzymes, expressed in recombinant forms, was carried out. Both enzymes could be purified as halo enzymes containing a FAD cofactor. Their thermostability was investigated and revealed that the Cm-BVMO is the most thermostable type I BVMO with an apparent melting temperature of 56 degrees C. Substrate profiling revealed that both eukaryotic BVMOs accept a wide range of ketones which include aromatic, aliphatic, aryl aliphatic and bicyclic ketones. In particular, linear aliphatic ketones (C9 and C12), carrying the keto functionality in different positions, resulted to be the best substrates in steady state kinetic analyses. In order to restore the BVMO-typifying sequence motif in the Pp-BVMO, a mutant was prepared (Y160H). Intriguingly, this mutation resulted in higher activities on most tested substrates. The recombinant enzymes displayed k(cat) values in the 0.1-0.2 s(-1) range, which is relatively low when compared with other known type I BVMOs. This may hint to a role in secondary metabolism in these photosynthetic organisms, though their exact function remains to be established. (C) 2013 Elsevier B.V. All rights reserved.

Within a newly identified set of flavin-containing monooxygenases (FMOs) from Rhodococcus jostii RHA1, we have identified three monooxygenases (FMO-E, FMO-F, and FMO-G) that are effective in catalyzing Baeyer-Villiger oxidations. These typeII FMOs display relaxed coenzyme specificity by accepting both NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) and NADH (reduced form of nicotinamide adenine dinucleotide), as a coenzyme, which is a novel and attractive feature among biocatalysts capable of conducting Baeyer-Villiger oxidations. We purified FMO-E and determined that the Michaelis constants for both coenzymes were in the micromolar range, whereas the activity was highest for NADH. By using the stopped-flow technique, formation of a peroxyflavin-enzyme intermediate was observed, which indicated that typeII FMOs follow a catalytic mechanism similar to that of other classB flavoprotein monooxygenases. A set of cyclobutanones and cyclohexanones were used to probe the regio- and enantioselectivity of all three recombinant monooxygenases. The biocatalysts readily accepted small cyclic ketones, which enabled the conversion of previously poorly accepted substrates by other monooxygenases (especially norcamphor), and exhibited excellent and unique regio- and enantioselectivities. Sequence analysis revealed that typeII FMOs that act as Baeyer-Villiger monooxygenases contain a unique N-terminal domain. Sequence conservation in this protein domain can be used to identify new NADH-dependent Baeyer-Villiger monooxygenases, which would facilitate future biocatalyst discovery efforts.

The precursor of the essential ether phospholipids is synthesized by a peroxisomal enzyme that uses a flavin cofactor to catalyze a reaction that does not alter the redox state of the substrates. The enzyme crystal structure reveals a V-shaped active site with a narrow constriction in front of the prosthetic group. Mutations causing inborn ether phospholipid deficiency, a very severe genetic disease, target residues that are part of the catalytic center. Biochemical analysis using substrate and flavin analogs, absorbance spectroscopy, mutagenesis, and mass spectrometry provide compelling evidence supporting an unusual mechanism of covalent catalysis. The flavin functions as a chemical trap that promotes exchange of an acyl with an alkyl group, generating the characteristic ether bond. Structural comparisons show that the covalent versus noncovalent mechanistic distinction in flavoenzyme catalysis and evolution relies on subtle factors rather than on gross modifications of the cofactor environment.

Cyclopentadecanone monooxygenase (CPDMO) is one of the latest additions to the established library of Baeyer-Villiger monooxygenases. Desymmetrizations of substituted cyclobutanones and -hexanones as well as kinetic resolutions of racemic cycloketones are efficiently catalyzed by CPDMO. Moreover the enzyme shows unprecedented preference in regiodivergent oxidations of terpenones and the bicyclic Geissman-Waiss lactone precursor giving access to the optical antipode of retronecine and other pyrrolizidine alkaloids. (C) 2011 Elsevier B.V. All rights reserved.

The applicability of Baeyer-Villiger monooxygenases (BVMOs) in organoboron chemistry has been explored through testing chemo-and enantioselective oxidations of a variety of boron-containing aromatic and vinylic compounds. Several BVMOs, namely: phenylacetone monooxygenase (PAMO), M446G PAMO mutant, 4-hydroxyacetophenone monooxygenase (HAPMO) and cyclohexanone monooxygenase (CHMO) were used in this study. The degree of chemoselectivity depends on the type of BVMO employed, in which the biocatalysts prefer boron-carbon oxidation over Baeyer-Villiger oxidation or epoxidation. Interestingly, it was discovered that PAMO can be used to perform kinetic resolution of boron-containing compounds with good enantioselectivities. These findings extend the known biocatalytic repertoire of BVMOs by showing a new family of compounds that can be oxidized by these enzymes.

A.R.-M. (FPU program) and I. L. (Ramón y Cajal Program) thank the Spanish MICINN for personal funding. A.C. thanks the Principado de Asturias for his predoctoral fellowship. Financial support from MICINN (Project CTQ2007-61126) is gratefully acknowledged. M.W.F. and G.d.G. receives support from the EU-FP7 “Oxygreen” project.

Baeyer-Villiger monooxygenases have been tested in the oxidation of racemic benzofused ketones. When employing a single Mutant of phenylacetone monooxygenase (M446G PAMO) under the proper reaction conditions, it was possible to achieve 3-substituted 3,4-dihydroisocoumarins with high yields and optical purities through regioselective dynamic kinetic resolution Processes.

Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S)-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S)-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S)-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS) based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

DyP-type peroxidases comprise a novel superfamily of heme-containing peroxidases which is unrelated to the superfamilies of known peroxidases and of which only a few members have been characterized in some detail. Here, we report the identification and characterization of a DyP-type peroxidase (TfuDyP) from the thermophilic actinomycete Thermobifida fusca. Biochemical characterization of the recombinant enzyme showed that it is a monomeric, heme-containing, thermostable, and Tat-dependently exported peroxidase. TfuDyP is not only active as dye-decolorizing peroxidase as it also accepts phenolic compounds and aromatic sulfides. In fact, it is able to catalyze enantioselective sulfoxidations, a type of reaction that has not been reported before for DyP-type peroxidases. Site-directed mutagenesis was used to determine the role of two conserved residues. D242 is crucial for catalysis while H338 represents the proximal heme ligand and is essential for heme incorporation. A genome database analysis revealed that DyP-type peroxidases are frequently found in bacterial genomes while they are extremely rare in other organisms. Most of the bacterial homologs are potential cytosolic enzymes, suggesting metabolic roles different from dye degradation. In conclusion, the detailed biochemical characterization reported here contributes significantly to our understanding of these enzymes and further emphasizes their biotechnological potential.

The application of three BVMOs for the enantioselective oxidation of 3-phenylbutan-2-ones with different substituents in the aromatic moiety is described. By choosing the appropriate biocatalyst and substrate combination, chiral ketones and esters can be obtained with excellent enantiopurities. This methodology could also be applied to the resolution of racemic alpha-alkyl benzylketones with longer alkyl chains as well as with two substituted alpha-substituted benzylacetones. A kinetic analysis revealed that the BVMOs studied effectively convert all tested compounds showing that the enzymes are tolerant towards the substrate structure while being highly enantioselective. These properties render BVMOs as valuable biocatalysts for the preparation of compounds with high interest in organic synthesis. (C) 2009 Elsevier Ltd. All rights reserved.