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Site-specific protein labeling methods are highly valuable tools for research and applications. We present a new protein labeling method that allows covalent attachment of a chromo-and fluorogenic flavin (FMN) to any targeted protein using a short flavinylation peptide-Tag. We show that this peptide can be as short as 7 residues and can be located at the N-Terminus, C-Terminus, or in internal regions of the target protein. Analogous to kinase-catalyzed phosphorylation, the flavin is covalently attached via a stable phosphothreonyl linkage. The site-specific covalent tethering of FMN is accomplished by using a bacterial flavin transferase. The covalent coupling of FMN was shown to work in Escherichia coli and Saccharomyces cerevisiae cells and could be performed in vitro, rendering the "Flavin-Tag"method a powerful tool for the selective decoration of proteins with a biocompatible redox-Active fluorescent chromophore.

Flavin as a cofactor is an extremely versatile molecule that participates in a wide range of biochemical reactions. A special characteristic of the flavin cofactor, unique for a metal free cofactor, is its ability to react in its reduced form with molecular oxygen. This is exploited in flavoprotein oxidases and monooxygenases. The flavin-mediated reduction of dioxygen can lead to various reactive oxygen species (ROS), primarily hydrogen peroxide and superoxide. No systematic analysis of the formation of such reduced oxygen species produced by flavoprotein oxidases and monooxygenases had been performed before. In this work, we investigated which ROS are formed by several prototype flavoprotein oxidases and monooxygenases: Phenylacetone monooxygenase (TfPAMO), an engineered TfPAMO variant that acts as an NADPH oxidase (TfPAMO C65D), eugenol oxidase (RjEUGO), and 5-hydroxymethyl furfural oxidase (MHMFO). The formed amounts of superoxide and hydrogen peroxide were measured under various conditions (different substrate concentrations, pH values and cosolvents). Interestingly, all flavoenzymes were found to produce, except for hydrogen peroxide, significant amounts of superoxide. Moreover, increased superoxide levels were measured at higher pH, which could be indicative for a pH-sensitive caged radical pair dissociation. To probe the effect of ROS formation on biocatalytic performance, conversions catalyzed by TfPAMO or RjEUGO, with or without catalase, were monitored. This revealed that catalase has a beneficial effect. No detrimental effect of the accumulation of superoxide on biocatalysis could be demonstrated. The results reveal that formation of ROS by flavoenzymes is highly dependent on the experimental conditions used. The results provide a better insight into the mechanism by which ROS is formed in flavoenzymes and may help studies or applications in which ROS formation should be promoted or minimized for industrial biocatalysis.

Dye‐decolorizing peroxidases (DyPs) constitute a superfamily of heme‐containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A‐C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D‐type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D‐type DyP ancestor, AncDyPD‐b1. Expression of AncDyPD‐b1 in Escherichia coli results in large amounts of a heme‐containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV‐Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady‐state kinetics and the crystal structure, reveal fine pH‐dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long‐range electron transfer process. Database Structural data are available in the PDB database under the accession number 7ANV., In this study, ancestral sequence reconstruction was performed to resurrect an ancestral fungal DyP‐type peroxidase, AncDyPD‐b1. The peroxidase could be overexpressed in Escherichia coli resulting in large amounts of a soluble, heme‐containing, and active enzyme. Combining site‐directed mutagenesis with UV‐Vis spectroscopy, resonance Raman spectroscopy, steady‐state kinetic analyses, and crystal structure elucidation, the roles of key active site residues were investigated.

Effective procedures for the synthesis of optically pure alcohols are highly valuable. A commonly employed method involves the biocatalytic reduction of prochiral ketones. This is typically achieved by using nicotinamide cofactor‐dependent reductases. In this work, we demonstrate that a rather unexplored class of enzymes can also be used for this. We used an F420‐dependent alcohol dehydrogenase (ADF) from Methanoculleus thermophilicus that was found to reduce various ketones to enantiopure alcohols. The respective (S) alcohols were obtained in excellent enantiopurity (>99 % ee). Furthermore, we discovered that the deazaflavoenzyme can be used as a self‐sufficient system by merely using a sacrificial cosubstrate (isopropanol) and a catalytic amount of cofactor F420 or the unnatural cofactor FOP to achieve full conversion. This study reveals that deazaflavoenzymes complement the biocatalytic toolbox for enantioselective ketone reductions., An alternative to NADH dependence: An F420‐dependent alcohol dehydrogenase can be used to asymmetrically reduce prochiral ketones. Conveniently, using a cheap sacrificial cosubstrate (isopropanol), the same enzyme can regenerate the deazaflavin cofactor.

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%.

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.

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-D-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme.

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.

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.

The vanillyl-alcohol oxidase (VAO) family is a rich source of biocatalysts for the oxidative bioconversion of phenolic compounds. Through genome mining and sequence comparisons, we found that several family members lack a generally conserved catalytic aspartate. This finding led us to study a VAO-homolog featuring a glutamate residue in place of the common aspartate. This 4-ethylphenol oxidase from Gulosibacter chungangensis (Gc4EO) shares 42 % sequence identity with VAO from Penicillium simplicissimum, contains the same 8 alpha-N-3-histidyl-bound FAD and uses oxygen as electron acceptor. However, Gc4EO features a distinct substrate scope and product specificity as it is primarily effective in the dehydrogenation of para-substituted phenols with little generation of hydroxylated products. The three-dimensional structure shows that the characteristic glutamate side chain creates a closely packed environment that may limit water accessibility and thereby protect from hydroxylation. With its high thermal stability, well defined structural properties and high expression yields, Gc4EO may become a catalyst of choice for the specific dehydrogenation of phenolic compounds bearing small substituents.

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.

With the aim of applying redox-neutral cascade reactions in organic media, fusions of a type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes. The lyophilized cell-free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane-1,4-diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert-butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ-butyrolactone, resulting in about 27 % analytical yield.

To expand the arsenal of industrially applicable oxidative enzymes, fusions of alcohol dehydrogenases with an NADPH‐oxidase were designed. Three different alcohol dehydrogenases (LbADH, TbADH, ADHA) were expressed with a thermostable NADPH‐oxidase fusion partner (PAMO C65D) and purified. The resulting bifunctional biocatalysts retained the catalytic properties of the individual enzymes, and acted essentially like alcohol oxidases: transforming alcohols to ketones by using dioxygen as mild oxidant, while merely requiring a catalytic amount of NADP+. In small‐scale reactions, the purified fusion enzymes show good performances, with 69–99 % conversion, 99 % ee with a racemic substrate, and high cofactor and enzyme total turnover numbers. As the fusion enzymes essentially act as oxidases, we found that commonly used high‐throughput oxidase‐activity screening methods can be used. Therefore, if needed, the fusion enzymes could be easily engineered to tune their properties., Going self‐sufficient: Alcohol dehydrogenases (ADHs) can catalyze selective alcohol oxidations and can be applied to kinetically resolve racemic alcohols, or to produce carbonyl building blocks. By fusing ADHs with an NADPH oxidase, self‐sufficient biocatalysts can be produced that act like alcohol oxidases. These enzyme fusions can be used for conversions and for activity screening to aid enzyme engineering.

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.

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.

Biocatalysis is increasingly used for synthetic purposes in the chemical and especially the pharmaceutical industry. Enzyme discovery and optimization which is frequently needed to improve biocatalytic performance rely on high-throughput methods for activity determination. These methods should ideally be generic and applicable to entire enzyme families. Hydrogen peroxide (H2O2) is a product of several biocatalytic oxidations and its formation can serve as a proxy for oxidative activity. We designed a genetically encoded sensor for activity measurement of oxidative biocatalysts via the amount of intracellularly-formed H2O2. A key component of the sensor is an H2O2-sensitive transcriptional regulator, OxyR, which is used to control the expression levels of fluorescent proteins. We employed the OxyR sensor to monitor the oxidation of glycerol to glyceraldehyde and of toluene to o-cresol catalysed by recombinant E. coli expressing an alcohol oxidase and a P450 monooxygenase, respectively. In case of the P450 BM3-catalysed reaction, we additionally monitored o-cresol formation via a second genetically encoded sensor based on the phenol-sensitive transcriptional activator, DmpR, and an orthogonal fluorescent reporter protein. Single round screens of mutant libraries by flow cytometry or by visual inspection of colonies on agar plates yielded significantly improved oxidase and oxygenase variants thus exemplifying the suitability of the sensor system to accurately assess whole-cell oxidations in a high-throughput manner., Chemical Science, 12 (44), ISSN:2041-6520, ISSN:2041-6539

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.

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.

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.

A set of bifunctional oxidase–peroxidases has been prepared by fusing four distinct oxidases to a peroxidase. Although such fusion enzymes have not been observed in nature, they could be expressed and purified in good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. Peroxidase fusions of alditol oxidase and chitooligosaccharide oxidase could be used for the selective detection of xylitol and cellobiose with a detection limit in the low‐micromolar range. The peroxidase fusions of eugenol oxidase and 5‐hydroxymethylfurfural oxidase could be used for dioxygen‐driven, one‐pot, two‐step cascade reactions to convert vanillyl alcohol into divanillin and eugenol into lignin oligomers. The designed oxidase–peroxidase fusions represent attractive biocatalysts that allow efficient biocatalytic cascade oxidations that only require molecular oxygen as an oxidant.

The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP+ to oxidize cyclohexanol to form cyclohexanone and NADPH. Both products are then used by CHMO to produce ε-caprolactone. In this study, these two redox-complementary enzymes were fused, to create a self-sufficient bifunctional enzyme that can convert alcohols to esters or lactones. Three different ADH genes were fused to a gene coding for a thermostable CHMO, in both orientations (ADH-CHMO and CHMO-ADH). All six fusion enzymes could be produced and purified. For two of the three ADHs, we found a clear difference between the two orientations: one that showed the expected ADH activity, and one that showed low to no activity. The ADH activity of each fusion enzyme correlated with its oligomerization state. All fusions retained CHMO activity, and stability was hardly affected. The TbADH-TmCHMO fusion was selected to perform a cascade reaction, producing ε-caprolactone from cyclohexanol. By circumventing substrate and product inhibition, a > 99% conversion of 200 mM cyclohexanol could be achieved in 24 h, with > 13,000 turnovers per fusion enzyme molecule. Electronic supplementary material The online version of this article (10.1007/s00253-017-8501-4) contains supplementary material, which is available to authorized users.

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

Synthetic lignin was prepared biocatalytically in a one-pot, two-step reaction using an oxidase/peroxidase cascade enzyme system. Using eugenol in combination with eugenol oxidase and a peroxidase, lignin-like material was produced. The cascade reaction takes advantage of the ability of the oxidase to produce coniferyl alcohol and hydrogen peroxide from eugenol and molecular oxygen. The hydrogen peroxide is used by the peroxidase for the formation of crosslinks that typify lignin. As eugenol oxidase has a broad substrate acceptance profile, also 4-allylphenol (chavicol) and 4-allyl-2,6-dimethoxyphenol could be used as precursors of the synthetic lignin. As a result, all three naturally occurring monolignols could be prepared and incorporated in the synthetic lignin. The reaction was optimized in order to achieve the highest possible yield of insoluble lignin oligomers and scaled up to 1 gram. Analysis of the water-insoluble product by gel permeation chromatography revealed the formation of relatively small lignin oligomers (approximate to 1000dalton). By using two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (2D HSQC NMR) analysis it could be demonstrated that the material contained alpha-O-4/-O-4, beta-O-4, beta-beta, beta-5 linkages and dibenzodioxocin units. All these features indicate that the biocatalytically produced material closely resembles natural lignin. While 54% of eugenol was converted into water-insoluble oligomers, the remaining substrate was converted into water-soluble dimers and tetramers which are important lignin model compounds. Therefore, the presented method represents a valuable and facile biocatalytic approach for the preparation of lignin-like material and potentially valuable chemicals.

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation.

A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP+ binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate. The resulting libraries were screened using whole cell biotransformations, and headspace gas chromatography-mass spectrometry was used to identify improved AcCHMO variants. This revealed that the I491A AcCHMO mutant exhibits a significant improvement over the wild type enzyme in the desired regioselectivity using 2-butanone as a substrate (40% vs. 26% methyl propanoate, respectively). Another interesting mutant is T56S AcCHMO, which exhibits a higher conversion yield (92%) and kcat (0.5 s-1) than wild type AcCHMO (52% and 0.3 s-1, respectively). Interestingly, the uncoupling rate for T56S AcCHMO is also significantly lower than that for the wild type enzyme. The T56S/I491A double mutant combined the beneficial effects of both mutations leading to higher conversion and improved regioselectivity. This study shows that even for a relatively small aliphatic substrate (2-butanone), catalytic efficiency and regioselectivity can be tuned by structure-inspired enzyme engineering.

In this proof of concept study we demonstrate direct utilization of limonene containing waste product orange peel as starting material for a biocatalytic cascade reaction. The product of this cascade is chiral carvolactone, a promising building block for thermoplastic polymers. Four different concepts were applied to augment limonene availability based on either water extraction solely, addition of extraction enhancers or biomass dissolution.

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.

By genome sequencing and analysis, we have been able to identify a gene cluster encoding a four component oxygenase which is able to oxidize D-limonene, L-limonene, cumene, and indole. Heterologous expression of the complete oxygenase system in Pseudomonas putida S12 allowed efficient and highly regiospecific biotransformation of D-limonene into (+)-trans-carveol. The discovered oxygenase represents a promising biocatalyst for the biotechnological valorisation of D-limonene. (C) 2015 Elsevier Ltd. All rights reserved.

Dye-decolorizing peroxidases (DyPs) represent a new class of oxidative enzymes for which the natural substrates are largely unknown. To explore the biocatalytic potential of a DyP, we have studied the substrate acceptance profile of a robust DyP peroxidase, a DyP from Thermobifida fusca (TfuDyP). While previous work established that this bacterial peroxidase is able to act on a few reactive dyes and aromatic sulfides, this work significantly expands its substrate scope towards lignin related compounds, flavors, and various dyes. (C) 2016 Elsevier Ltd. All rights reserved.

By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1.

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.

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

Thermobifida fusca is a well-known cellulose-degrading actinomycete, which produces various glycoside hydrolases for this purpose. However, despite the presence of putative chitinase genes in its genome, T. fusca has not been reported to grow on chitin as sole carbon source. In this study, a gene encoding a putative membrane anchored GH18 chitinase (Tfu0868) from T. fusca has been cloned and overexpressed in Escherichia coli. The protein was produced as SUMO fusion protein and, upon removal of the SUMO domain, soluble pure TfChi18A was obtained with yields typically amounting to 150 mg per litre of culture. The enzyme was found to be relatively thermostable (apparent T-m = 57.5 degrees C) but not particularly thermoactive, the optimum temperature being 40-45 degrees C. TfChi18A bound to alpha- and beta-chitin and degraded both these substrates. Interestingly, activity towards colloidal chitin was minimal and in this case, substrate inhibition was observed. TfChi18A also cleaved soluble chitooligosaccharides and showed a clear preference for substrates having five sugars or more. While these results show that TfChi18A is a catalytically competent GH18 chitinase, the observed catalytic rates were low compared to those of well-studied GH18 chitinases. This suggests that TfChi18A is not a true chitinase and not likely to endow T. fusca with the ability to grow on chitin. (C) 2016 Elsevier B.V. All rights reserved.

Mammalian flavin-containing monooxygenases, which are difficult to obtain and study, play a major role in detoxifying various xenobiotics. To provide alternative biocatalytic tools to generate flavin-containing monooxygenases (FMO)-derived drug metabolites, a collection of microbial flavoprotein monooxygenases, sequence-related to human FMOs, was tested for their ability to oxidize a set of xenobiotic compounds. For all tested xenobiotics [nicotine, lidocaine, 3-(methylthio)aniline, albendazole, and fenbendazole], one or more monooxygenases were identified capable of converting the target compound. Chiral liquid chromatography with tandem mass spectrometry analyses of the conversions of 3-(methylthio)aniline, albendazole, and fenbendazole revealed that the respective sulfoxides are formed in good to excellent enantiomeric excess (e.e.) by several of the tested monooxygenases. Intriguingly, depending on the chosen microbial monooxygenase, either the (R)- or (S)-sulfoxide was formed. For example, when using a monooxygenase from Rhodococcus jostii the (S)-sulfoxide of albendazole (ricobendazole) was obtained with a 95% e.e. whereas a fungal monooxygenase yielded the respective (R)-sulfoxide in 57% e.e. For nicotine and lidocaine, monooxygenases could be identified that convert the amines into their respective N-oxides. This study shows that recombinantly expressed microbial monooxygenases represent a valuable toolbox of mammalian FMO mimics that can be exploited for the production of FMO-associated xenobiotic metabolites.

A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 degrees C) when compared to PAMO in solution (no activity detected after 1 h at 60 degrees C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier. (C) 2015 Elsevier Inc. All rights reserved.

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.

The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615) and XP_003665713 (MtVAO713), were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate oxidases (glucooligosaccharide oxidase from Acremonium strictum and lactose oxidase from Microdochium nivale) eleven mutants were designed to redirect the catalytic scope of ChitO for improved oxidation of lactose, cellobiose and maltose. The catalytic properties of the most interesting mutants were further improved by combining single mutations. This has resulted in the creation of a set of ChitO variants that display totally different substrate tolerances. One ChitO variant shows a dramatic improvement in catalytic efficiency towards oxidation of glucose, cellobiose, lactose, and maltose. We also describe a ChitO variant with the highest catalytic efficiency in GlcNAc oxidation so far reported in the literature.

Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 A degrees C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k (obs) of nearly 1000 s(-1) for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 A degrees C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities.

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.

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.).

The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass polysaccharides. At the same time basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Here we describe a method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity. The method was used to quantify the activity of a four-domain LPMO from Vibrio cholerae, GbpA, which is a virulence factor with no obvious role in biomass processing. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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.

Highlights • Cyclohexanone monooxygenase was stabilized by an in silico designed disulfide bond. • Stabilizing disulfide bonds were successfully designed based on a model structure. • The half-life at 30 °C was increased 12-fold for the mutant enzyme. • The apparent melting point was increased by 6 °C for the mutant enzyme. • The most stabilizing disulfide bond spans only one residue., Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer–Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C–A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.

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.

DyP peroxidases comprise a novel superfamily of heme-containing peroxidases, which is unrelated to the superfamilies of plant and animal peroxidases. These enzymes have so far been identified in the genomes of fungi, bacteria, as well as archaea, although their physiological function is still unclear. DyPs are bifunctional enzymes displaying not only oxidative activity but also hydrolytic activity. Moreover, these enzymes are able to oxidize a variety of organic compounds of which some are poorly converted by established peroxidases, including dyes, β-carotene, and aromatic sulfides. Interestingly, accumulating evidence shows that microbial DyP peroxidases play a key role in the degradation of lignin. Owing to their unique properties, these enzymes are potentially interesting for a variety of biocatalytic applications. In this review, we deal with the biochemical and structural features of DyP-type peroxidases as well as their promising biotechnological potential.

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.