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Silybin and isosilybin are the major active components of silymarin, which have long been used to treat liver disease. Market available silymarin is currently extracted from the seed of milk thistle (Silybum marianum Linn. Gaertn) as a mixture, which consists of at least 7 flavonolignans including silybin and isosilybin, and other flavonoids. Plant extraction suffers from limited yield, geographical variations and market dynamics due to insufficient supply and inconsistent product purity. There is a pressing need to develop cost-efficient processes to manufacture silybin-based drugs. Silybin and isosilybin biosynthesis involves the oxidation of the expensive precursor coniferyl alcohol by milk thistle ascorbate peroxidase (APX1). However, the limited supply of coniferyl alcohol restricts the efficient production of silymarin-based drugs. In this study, we seek to develop alternative routes for the synthesis of silybin and isosilybin. Enzymology studies led us to identify that APX1 was not stereoselective and eugenol could be used as a surrogate substrate for silybin production. We then established an enzymatic cascade, which consists of Penicillium simplicissimum vanillyl alcohol oxidase (PsVAO) and APX1, for the efficient conversion of eugenol and taxifolin to silybin and isosilybin. Using H2O2 as the electron acceptor, APX1 converts coniferyl alcohol and taxifolin to silybin, and detoxifies the byproduct H2O2 at the same time. With cofactor heme enhancement, reaction condition optimization and scale-up, our enzymatic cascades yielded 2.58 g L−1 silybin and isosilybin. This efficient enzymatic cascade represents the first route to produce silybin and isosilybin by microorganisms. The coupling of PsVAO with APX1, and the detoxification of H2O2 by APX1 also offers a promising approach to generate silybin-based drugs.

Vanillin is a natural flavoring agent that is widely used in the bioengineering industry. To enable sustainable development, joint consideration of bacterial performance and negative environmental impacts are critical to vanillin biosynthesis. In this study, a cold shock protein (csp) gene was upregulated for maintaining stable growth in Arthrobacter sp. C2 responding to vanillin and cold stress. Furthermore, the recombinant strain C2 was constructed by simultaneously deleting the xylC gene encoding benzaldehyde dehydrase and overexpressing the pchF gene encoding vanillyl alcohol oxidase and achieved a maximum vanillin productivity of 0.85 mg/g DCW/h with alkaline lignin as the substrate. Finally, this process generated an environmental impact value of 25.05, which was the lowest environmental impact achieved according to life cycle assessment (LCA). Improvement strategies included reducing electricity consumption and replacing chemicals. This study achieved the development of an effective strategy, and future studies should focus on precise vanillin biosynthesis methods for large-scale application.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a K(m) of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes. IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β′ linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

This review presents a historical outline of the research on vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum, one of the canonical members of the VAO/PCMH flavoprotein family. After describing its discovery and initial biochemical characterization, we discuss the physiological role, substrate scope, and catalytic mechanism of VAO, and review its three-dimensional structure and mechanism of covalent flavinylation. We also explain how protein engineering provided a deeper insight into the role of certain amino acid residues in determining the substrate specificity and enantioselectivity of the enzyme. Finally, we summarize recent computational studies about the migration of substrates and products through the enzyme's structure and the phylogenetic distribution of VAO and related enzymes.

Coniferyl alcohol is a valuable chemical. However, the current approaches to obtain coniferyl alcohol are either unefficient or expensive. Penicillium simplicissimum vanillyl alcohol oxidase (PsVAO) can be used to produce coniferyl alcohol. However, PsVAO intrinsically produces harmful byproduct H2O2. Utilizing catalase to decompose H2O2 is a potential straightforward approach; however, catalase can also exhibit peroxidase activity to facilitate coniferyl alcohol over-oxidation. In this study, catalases exhibiting both high catalase activity and low peroxidase activity were found out, and introduced into the bioconversion systems. Our results showed that eliminating H2O2 in situ released H2O2 inhibition of PsVAO, improved coniferyl alcohol production and eliminated coniferyl alcohol over-oxidation. Finally, coniferyl alcohol titer, molar yield, and productivity reached 22.9 g/L, 78.7%, and 0.5 g/(L × h) respectively. An efficient coniferyl alcohol production method was developed by overcoming the intrinsic disadvantages of PsVAO.

Over time, organisms have evolved strategies to cope with the abundance of dioxygen on Earth. Oxygen-utilizing enzymes tightly control the reactions involving O-2 mostly by modulating the reactivity of their cofactors. Flavins are extremely versatile cofactors that are capable of undergoing redox reactions by accepting either one electron or two electrons, alternating between the oxidized and the reduced states. The physical and chemical principles of flavin-based chemistry have been investigated widely. In the following pages we summarize the state of the art on a key area of research in flavin enzymology: the molecular basis for the activation of O-2 by flavin-dependent oxidases and monooxygenases. In general terms, oxidases use O-2 as an electron acceptor to produce H2O2, while monooxygenases activate O-2 by forming a flavin intermediate and insert an oxygen atom into the substrate. First, we analyze how O-2 reaches the flavin cofactor embedded in the protein matrix through dedicated access pathways. Then we approach O-2 activation from the perspective of the monooxygenases, their preferred intermediate, the C(4a)-(hydro)peroxyflavin, and the cases in which other intermediates have been described. Finally, we focus on understanding how the architectures developed in the active sites of oxidases promote O-2 activation and which other factors operate in its reactivity.

The degradation of lignin is the main rate-limiting step in the bio-pulping of rice straw. A lignin-degrading bacterial consortium LDC, which can efficiently degrade lignin of reed, was screened in the early stage of our laboratory work. In present study, 7-day incubation of LDC can degrade rice straw lignin by 31.18% in mineral salt medium. The communities’ structure of different incubation phases varied greatly, in which high abundance (44.78%) of Anaerocolumna was first found. The expression levels of lignin degradation enzyme class II peroxidase (AA2), vanillyl alcohol oxidase (AA4) and 1,4-benzoquinone reductase (AA6) during peak phase (48 h) were significantly up-regulated than initial phase (24 h), increasing by 112%, 165% and 67%, respectively, and 42.86% AA2 was from Thaurea; 100% AA4 was from Clostridium; 62.5% AA6 was from Pseudomonas. These provide microbial resources and data support for the industrialization of rice straw bio-pulping.

The VAO/PCMH flavoprotein family consists of structurally homologous flavin-dependent enzymes that catalyze a wide range of chemical reactions. Family members share an architecture consisting of a conserved FAD-binding domain and a more variable substrate-binding domain, which enables varying interactions with a range of substrates while maintaining the same cofactor-binding fold. Here, we provide an overview of the current state of our knowledge on the members of the VAO/PCMH family. Based on a phylogenetic analysis, we divide the family into 11 subgroups. We discuss the properties of these subgroups, focusing on recent developments in terms of the discovery of new family members and mechanistic advances. We also highlight open questions that will provide challenges for future research.

Pinoresinol is a natural lignan with a high market value that has potential pharmacological and food supplement applications. Pinoresinol is currently isolated from plants, which suffers from low efficiency and yield. To produce pinoresinol from inexpensive and industrially available eugenol, an in vivo enzymatic cascade composed of vanillyl alcohol oxidase and peroxidase was designed, which scavenges H2O2 automatically and eliminates protein purification and cofactor addition. Two peroxidases were screened and identified from Escherichia coli BL21 (DE3), and tested in the enzymatic cascade. To balance the flux, different genetic architectures were constructed by using ePathBrick and fusion gene approaches. Scavenging H2O2 alleviated by-product toxicity and enzyme inhibition, and led to efficient pinoresinol production. Optimization of the reaction conditions achieved a titer of 11.29 g/L pinoresinol. The molar yield and productivity were 52.77% and 1.03 g/(L × h), respectively. The elegant strategy developed herein utilizes the harmful by-product to drive the biosynthetic reaction forward and simultaneously detoxify cells, thereby preventing enzyme inhibition. Biotechnol. Bioeng. 2017;114: 2066–2074. © 2017 Wiley Periodicals, Inc.

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.

The flavoprotein d-6-hydroxynicotine oxidase catalyzes an early step in the oxidation of (R)-nicotine, the oxidation of a carbon–nitrogen bond in the pyrrolidine ring of (R)-6-hydroxynicotine. The enzyme is a member of the vanillyl alcohol oxidase/p-cresol methylhydroxylase family of flavoproteins. The effects of substrate modifications on the steady-state and rapid-reaction kinetic parameters are not consistent with the quinone-methide mechanism of p-cresol methylhydroxylase. There is no solvent isotope effect on the k(cat)/K(amine) value with either (R)-6-hydroxynicotine or the slower substrate (R)-6-hydroxynornicotine. The effect of pH on the rapid-reaction kinetic parameters establishes that only the neutral form of the substrate and the correctly protonated form of the enzyme bind. The active-site residues Lys348, Glu350, and Glu352 are all properly positioned for substrate binding. The K348M substitution has only a small effect on the kinetic parameters; the E350A and E350Q substitutions decrease the k(cat)/K(amine) value by ~20- and ~220-fold, respectively, and the E352Q substitution decreases this parameter ~3800-fold. The k(cat)/K(amine)–pH profile is bell-shaped. The pK(a) values in that profile are altered by replacement of (R)-6-hydroxynicotine with (R)-6-hydroxynornicotine as the substrate and by the substitutions for Glu350 and Glu352, although the profiles remain bell-shaped. The results are consistent with a network of hydrogen-bonded residues in the active site being involved in binding the neutral form of the amine substrate, followed by the transfer of a hydride from the amine to the flavin.

Coniferyl alcohol is a common monolignol, which is a precursor of lignin. Coniferyl alcohol is rarely found as free molecules in plants based on its conversion to lignins by peroxidases or laccases. It can be used for biosynthesis of silybin and a lot of pinoresinol derivatives. Although increased output of coniferyl alcohol has been attempted, few methods are effective. In this study, we evaluated 20 promoters for their ability to express genes in Penicillium simplicissimum encoding vanillyl alcohol oxidase (PsVAO) and catalase (ScCTA1) for producing coniferyl alcohol, while eliminating harmful H2O2. We constructed 100 combinations of constitutive promoters in E. coli to enhance gene expression. Then orthogonal experiments, fed-batch kinetic model and CRISPR/Cas9 system were followed to improve the yield of coniferyl alcohol, while eliminate the pinoresinol. Finally, the coniferyl alcohol titers, bioconversion rates, and productivity levels reaching 53.90 g/L, 86.72 %, and 2.45 g/(L × h), respectively.

Flavoenzymes are versatile catalysts that mostly facilitate redox reactions such as the oxygenation of organic substrates. Commonly, flavin monooxygenases employ a flavin-C4a-(hydro)peroxide as oxygenating species. Recently, however, a modified N5-functionalized flavin cofactor featuring a distinct nitrone moiety — the flavin-N5-oxide — was reported for the first time as oxygenating species in the bacterial enzyme EncM that catalyzes the dual oxidation of a reactive poly-β-ketone substrate. Meanwhile, additional flavoenzymes have been reported that form the flavin-N5-oxide. Here, we highlight aspects of the discovery and characterization of this novel flavin redox state with a focus on recent findings that shed more light onto its chemical features and enzymatic formation. We furthermore provide a rationale for the oxygenase functionality of EncM by contrast with structurally related flavin oxidases and dehydrogenases from the vanillyl alcohol oxidase/p-cresol methylhydroxylase flavoprotein (VAO/PCMH) superfamily. In addition, the possible biological roles of the flavin-N5-oxide are discussed.

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

The enantioselective oxyfunctionalisation of C−H bonds is a highly interesting reaction, as it provides access to chiral alcohols that are important pharmaceutical building blocks. However, it is hard to achieve using traditional methods. One way in which it can be achieved is through the action of oxidative enzymes. Although many reports of the oxyfunctionalisation capabilities of enzymes at an analytical scale have been published, reports on the use of enzymes to achieve oxyfunctionalisation on a synthetically relevant scale are fewer. Here, we describe the scale-up of the conversion of 4-ethylphenol to (R)-1-(4′-hydroxyphenyl)ethanol using the flavin-dependent enzyme vanillyl alcohol oxidase. The process was optimised by testing different reaction media and substrate and enzyme concentrations and by performing it under an oxygen atmosphere. Under optimised reaction conditions, 4.10 g (R)-1-(4′-hydroxyphenyl)ethanol at 97% ee was obtained from 10 g 4-ethylphenol (isolated yield 36%). These results highlight some of the challenges that can be encountered during scale-up of an enzymatic oxyfunctionalisation process to a synthetically relevant scale and will be of use for the development of enzymatic processes for the synthesis of industrially relevant compounds. (Figure presented.).

Over time, organisms have evolved strategies to cope with the abundance of dioxygen on Earth. Oxygen-utilizing enzymes tightly control the reactions involving O-2 mostly by modulating the reactivity of their cofactors. Flavins are extremely versatile cofactors that are capable of undergoing redox reactions by accepting either one electron or two electrons, alternating between the oxidized and the reduced states. The physical and chemical principles of flavin-based chemistry have been investigated widely. In the following pages we summarize the state of the art on a key area of research in flavin enzymology: the molecular basis for the activation of O-2 by flavin-dependent oxidases and monooxygenases. In general terms, oxidases use O-2 as an electron acceptor to produce H2O2, while monooxygenases activate O-2 by forming a flavin intermediate and insert an oxygen atom into the substrate. First, we analyze how O-2 reaches the flavin cofactor embedded in the protein matrix through dedicated access pathways. Then we approach O-2 activation from the perspective of the monooxygenases, their preferred intermediate, the C(4a)-(hydro)peroxyflavin, and the cases in which other intermediates have been described. Finally, we focus on understanding how the architectures developed in the active sites of oxidases promote O-2 activation and which other factors operate in its reactivity.

Vanillyl alcohol oxidase can make vanillin, the major flavour component of vanilla. We have studied this enzyme in great detail to better understand how it works and how exactly it produces vanillin. We have identified two different paths that guide substrates to where the reaction takes place. Once there, some substrates form an adduct, preventing product formation. Based on our work, we predict that the size of the substrate determines if the adduct is formed, and that one amino acid in the active centre of the enzyme is crucial for this process. Our work will help other researchers to improve this enzyme so that it can make vanillin more efficiently.

The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a ‘gatekeeper’ amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60 000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the ‘oxyanion hole’ in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. Database Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF .

Vanillyl alcohol oxidase (VAO) is a homo-octameric flavoenzyme belonging to the VAO/PCMH family. Each VAO subunit consists of two domains, the FAD-binding and the cap domain. VAO catalyses, among other reactions, the two-step conversion of p-creosol (2-methoxy-4-methylphenol) to vanillin (4-hydroxy-3-methoxybenzaldehyde). To elucidate how different ligands enter and exit the secluded active site, Monte Carlo based simulations have been performed. One entry/exit path via the subunit interface and two additional exit paths have been identified for phenolic ligands, all leading to the si side of FAD. We argue that the entry/exit path is the most probable route for these ligands. A fourth path leading to the re side of FAD has been found for the co-ligands dioxygen and hydrogen peroxide. Based on binding energies and on the behaviour of ligands in these four paths, we propose a sequence of events for ligand and co-ligand migration during catalysis. We have also identified two residues, His466 and Tyr503, which could act as concierges of the active site for phenolic ligands, as well as two other residues, Tyr51 and Tyr408, which could act as a gateway to the re side of FAD for dioxygen. Most of the residues in the four paths are also present in VAO’s closest relatives, eugenol oxidase and p-cresol methylhydroxylase. Key path residues show movements in our simulations that correspond well to conformations observed in crystal structures of these enzymes. Preservation of other path residues can be linked to the electron acceptor specificity and oligomerisation state of the three enzymes. This study is the first comprehensive overview of ligand and co-ligand migration in a member of the VAO/PCMH family, and provides a proof of concept for the use of an unbiased method to sample this process., Author summary Enzymes are bionanomachines, which speed up chemical reactions in organisms. To understand how they achieve that, we need to study their mechanisms. Computational enzymology can show us what happens in the enzyme’s active site during a reaction. But molecules need first to reach the active site before a reaction can start. The process of substrate entry and product exit to the active site is often neglected when studying enzymes. However, these two events are of fundamental importance to the proper functioning of any enzyme. We are interested in these dynamic processes to complete our understanding of the mode of action of enzymes. In our work, we have studied substrate and product migration in vanillyl alcohol oxidase. This enzyme can produce the flavour vanillin and enantiopure alcohols, but also catalyses other reactions. The named products are of interest to the flavour- and fine-chemical industries.

The VAO/PCMH flavoprotein family consists of structurally homologous flavin-dependent enzymes that catalyze a wide range of chemical reactions. Family members share an architecture consisting of a conserved FAD-binding domain and a more variable substrate-binding domain, which enables varying interactions with a range of substrates while maintaining the same cofactor-binding fold. Here, we provide an overview of the current state of our knowledge on the members of the VAO/PCMH family. Based on a phylogenetic analysis, we divide the family into 11 subgroups. We discuss the properties of these subgroups, focusing on recent developments in terms of the discovery of new family members and mechanistic advances. We also highlight open questions that will provide challenges for future research.

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 recently discovered class of bicovalent flavoproteins is an interesting group of enzymes because of their unusual cofactor binding mode, their open active sites and the bulky substrates they can accept. Through a sequence comparison study we have identified a conserved sequence region in bicovalent flavoproteins that is different from monocovalent flavoproteins. Based on this and the available structural information we have designed mutants of the prototype monocovalent flavoprotein, 6-hydroxy-D-nicotine oxidase (6HDNO), in order to introduce a second cofactor-protein linkage. Two amino acid replacements, namely histidine 130 to a cysteine and leucine 138 to a histidine, were sufficient to create a bicovalent 6HDNO. The introduced cysteine forms a covalent bond with FAD as found in natural bicovalent flavoproteins, while the second mutation was found to be essential to facilitate the formation of the cysteinyl linkage. This points to an important role of the introduced histidine in stabilizing a negative charge of the isoalloxazine ring during covalent flavinylation. The His130Cys/Leu138His 6HDNO is still active and shows a higher midpoint redox potential when compared to wild-type 6HDNO. This agrees well with the previous studies that have shown that bicovalent flavoenzymes have extremely high redox potentials (C) 2014 Elsevier Ltd. All rights reserved.

A novel approach for the synthesis of vanillin employing a three-step two-enzymatic cascade sequence is reported. Cytochrome P450 monooxygenases are known to catalyse the selective hydroxylation of aromatic compounds, which is one of the most challenging chemical reactions. A set of rationally designed variants of CYP102A1 (P450 BM3) from Bacillus megaterium at the amino acid positions 47, 51, 87, 328 and 437 was screened for conversion of the substrate 3-methylanisole to vanillyl alcohol via the intermediate product 4-methylguaiacol. Furthermore, a vanillyl alcohol oxidase (VAO) variant (F454Y) was selected as an alternative enzyme for the transformation of one of the intermediate compounds via vanillyl alcohol to vanillin. As a proof of concept, the bi-enzymatic three-step cascade conversion of 3-methylanisole to vanillin was successfully evaluated both in vitro and in vivo.

This review provides an overview of oxidases that utilise a flavin cofactor for catalysis. This class of oxidative flavoenzymes has shown to harbour a large number of biotechnologically interesting enzymes. Applications range from their use as biocatalysts for the synthesis of pharmaceutical compounds to the integration in biosensors. Through the recent developments in genome sequencing, the number of newly discovered oxidases is steadily growing. Recent progress in the field of flavoprotein oxidase discovery and the obtained biochemical knowledge on these enzymes are reviewed. Except for a structure-based classification of known flavoprotein oxidases, also their potential in recent biotechnological applications is discussed.

Background Pinoresinol is a high-value plant-derived lignan with multiple health supporting effects. Enantiomerically pure pinoresinol can be isolated from natural sources, but with low efficiency. Most chemical and biocatalytic approaches that have been described for the synthesis of pinoresinol furnish the racemic mixture. In this study we devised a three-step biocatalytic cascade for the production of enantiomerically pure pinoresinol from the cheap compound eugenol. Two consecutive oxidations of eugenol through vanillyl-alcohol oxidase and laccase are followed by kinetic resolution of racemic pinoresinol by enantiospecific pinoresinol reductases. Results The addition of the enantiospecific pinoresinol reductase from Arabidopsis thaliana for kinetic resolution of (±)-pinoresinol to an in vitro cascade involving the vanillyl-alcohol oxidase from Penicillium simplicissimum and the bacterial laccase CgL1 from Corynebacterium glutamicum resulted in increasing ee values for (+)-pinoresinol; however, an ee value of 34 % was achieved in the best case. The ee value could be increased up to ≥99 % by applying Escherichia coli-based whole-cell biocatalysts. The optimized process operated in a one-pot “two-cell” sequential mode and yielded 876 µM (+)-pinoresinol with an ee value of 98 %. Switching the reductase to the enantiospecific pinoresinol lariciresinol reductase from Forsythia intermedia enabled the production of 610 µM (−)-pinoresinol with an ee value of 97 %. Conclusion A new approach for the synthesis of enantiomerically pure (+)- and (−)-pinoresinol is described that combines three biotransformation steps in one pot. By switching the reductase in the last step, the whole-cell biocatalysts can be directed to produce either (+)- or (−)-pinoresinol. The products of the reductases’ activity, (−)-lariciresinol and (−)-secoisolariciresinol, are valuable precursors that can also be applied for the synthesis of further lignans. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0472-0) contains supplementary material, which is available to authorized users.

The VAO/PCMH family of flavoenzymes is a family of structurally related proteins that catalyse a wide range of oxidation reactions. It contains a subfamily of enzymes that catalyse the oxidation of para-substituted phenols using covalently bound FAD cofactors (the 4PO subfamily). This subfamily is composed of two oxidases, vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO), and two flavocytochrome dehydrogenases, para-cresol methylhydroxylase (PCMH) and eugenol hydroxylase (EUGH). Although they catalyse similar reactions, these enzymes differ in terms of their electron acceptor preference and oligomerization state. For example, VAO forms homo-octamers that can be described as tetramers of stable dimers, whereas EUGO is exclusively dimeric in solution. A possible explanation for this difference is the presence of a loop at the dimer–dimer interface in VAO that is not present in EUGO. Here, the role played by this loop in determining the quaternary structure of these enzymes is investigated. A VAO variant where the loop was deleted, loopless VAO, exclusively formed dimers. However, introduction of the loop into EUGO was not sufficient to induce its octamerization. Neither variant displayed major changes in its catalytic properties as compared to the wild-type enzyme. Bioinformatic analysis revealed that the presence of the loop is conserved within putative fungal oxidases of the 4PO subgroup, but it is never found in putative bacterial oxidases or dehydrogenases. Our results shed light on the molecular mechanism of homo-oligomerization of VAO and the importance of oligomerization for its enzymatic function. Enzymes p-cresol methylhydroxylase (4-methylphenol:acceptor oxidoreductase (methyl-hydroxylating), EC 1.17.99.1); vanillyl alcohol oxidase (vanillyl alcohol:oxygen oxidoreductase, EC 1.1.3.38).

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol—4‐(1‐hydroxyethyl)‐2‐methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent‐inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p‐substituents while tolerating o‐methoxy substituents.