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The F420 deazaflavin cofactor is an intriguing molecule as it structurally resembles the canonical flavin cofactor, although behaves as a nicotinamide cofactor due to its obligate hydride-transfer reactivity and similar low redox potential. Since its discovery, numerous enzymes relying on it have been described. The known deazaflavoproteins are taxonomically restricted to Archaea and Bacteria. The biochemistry of the deazaflavoenzymes is diverse and they exhibit great structural variability. In this study a thorough sequence and structural homology evolutionary analysis was performed in order to generate an overarching classification of the F420 -dependent oxidoreductases. Five different deazaflavoenzyme Classes (I-V) are described according to their structural folds as follows: Class I encompassing the TIM-barrel F420 -dependent enzymes; Class II including the Rossmann fold F420 -dependent enzymes; Class III comprising the β-roll F420 -dependent enzymes; Class IV which exclusively gathers the SH3 barrel F420 -dependent enzymes and Class V including the 3 layer ββα sandwich F420 -dependent enzymes. This classification provides a framework for the identification and biochemical characterization of novel deazaflavoenzymes. This article is protected by copyright. All rights reserved.

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.

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.

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.

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

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.

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

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

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

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.

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

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

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.

The F420 deazaflavin cofactor is an intriguing molecule as it structurally resembles the canonical flavin cofactor, although biochemically behaves as a nicotinamide cofactor. Since its discovery, numerous enzymes relying on it have been described. The known deazaflavoproteins are taxonomically restricted to Archaea and Bacteria. The biochemistry of the deazaflavoenzymes is diverse and they exhibit some degree of structural variability as well. In this study a thorough sequence and structural homology evolutionary analysis was performed in order to generate an overarching classification of all known F420-dependent oxidoreductases. Five different superfamilies are described: Superfamily I, TIM-barrel F420-dependent enzymes; Superfamily II, Rossmann fold F420-dependent enzymes; Superfamily III, β-roll F420-dependent enzymes; Superfamily IV, SH3 barrel F420-dependent enzymes and Superfamily V, 3 layer ββα sandwich F420-dependent enzymes. This classification aims to be the framework for the identification, the description and the understanding the biochemistry of novel deazaflavoenzymes.

In this paper, we report on the discovery of a novel, robust protein cage (encapsulin) that we could use for packaging various biocatalysts. We have elucidated the structure of the stable encapsulin by electron microscopy and X-ray diffraction. Furthermore, we developed an effective expression system for the encapsulin and a facile protocol for preparing encapsulated enzymes. By packaging and testing various enzymes (varying in size, oligomeric structure, and cofactor type) we demonstrate that, through encapsulation, the enzymes become significantly more stable. We also provide evidence that the pores of the encapsulin, through a size-exclusion effect, can modulate the substrate acceptance profile of an encapsulated enzyme.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (Delta T-m >= +3 degrees C). By combining mutations, a 10-fold mutant was generated with a T-m of 94 degrees C (+51 degrees C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a T-m of 88 degrees C (+45 degrees C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.

Type I Baeyer&ndash, Villiger monooxygenases (BVMOs) are flavin-dependent monooxygenases that catalyze the oxidation of ketones to esters or lactones, a reaction otherwise performed in chemical processes by employing hazardous and toxic peracids. Even though various BVMOs are extensively studied for their promising role in industrial biotechnology, there is still a demand for enzymes that are able to retain activity at high saline concentrations. To this aim, and based on comparative in silicoanalyses, we cloned HtBVMO from the extremely halophilic archaeon Haloterrigena turkmenica DSM 5511. When expressed in standard mesophilic cell factories, proteins adapted to hypersaline environments often behave similarly to intrinsically disordered polypeptides. Nevertheless, we managed to express HtBVMO in Escherichia coli and could purify it as active enzyme. The enzyme was characterized in terms of its salt-dependent activity and resistance to some water&ndash, organic-solvent mixtures. Although HtBVMO does not seem suitable for industrial applications, it provides a peculiar example of an alkalophilic and halophilic BVMO characterized by an extremely negative charge. Insights into the behavior and structural properties of such salt-requiring may contribute to more efficient strategies for engineering the tuned stability and solubility of existing BVMOs.

The conversion of a series of pharmaceutical compounds was examined with three variants of cytochrome P450BM3 fused to phosphite dehydrogenase (PTDH) to enable cofactor recycling. Conditions for enzyme production were optimized, and the purified PTDH-P450BM3 variants were tested against 32 commercial drugs by using rapid UPLC-MS analysis. The sets of mutations (R47L/F87V/L188Q and R47L/F87V/L188Q/E267V/G415S) improved conversion for all compounds, and a variety of products were detected. Product analysis showed that reaction types included C-hydroxylation, N-oxidation, demethylation, and aromatization. Interestingly, enzymatic aromatization could occur independent of the addition of reducing coenzyme. These results identified new conversions catalyzed by P450BM3 variants and showed that a small set of mutations in the oxygenase domain could broaden both substrate range and reaction type.

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

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

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.

F420H2-dependent enzymes reduce a wide range of substrates that are otherwise recalcitrant to enzyme-catalyzed reduction, and their potential for applications in biocatalysis has attracted increasingly attention. Thermobifida fusca is a moderately thermophilic bacterium and holds high biocatalytic potential as a source for several highly thermostable enzymes. We report here on the isolation and characterization of a thermostable F420:NADPH oxidoreductase (Tfu-FNO) from T. fusca, being the first F420-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in Escherichia coli, yielding up to 200 mg recombinant enzyme per liter of culture. We found that Tfu-FNO is highly thermostable, reaching its highest activity at 65 °C and that Tfu-FNO is likely to act in vivo as an F420 reductase at the expense of NADPH, similar to its counterpart in Streptomyces griseus We obtained the crystal structure of FNO in complex with NADP+ at 1.8 Å resolution, providing the first bacterial FNO structure. The overall architecture and NADP+-binding site of Tfu-FNO were highly similar to those of the Archaeoglobus fulgidus FNO (Af-FNO). The active site is located in a hydrophobic pocket between an N-terminal dinucleotide-binding domain and a smaller C-terminal do-main. Residues interacting with the 2'-phosphate of NADP+ were probed by targeted mutagenesis, indicating that Thr28, Ser50, Arg51, and Arg55 are important for discriminating between NADP+ and NAD+. Interestingly, a T28A mutant increased the kinetic efficiency more than three-fold as compared with the wild-type enzyme when NADH is the substrate. The biochemical and structural data presented here provide crucial insights into the molecular recognition of the two cofactors, F420 and NAD(P)H by FNO.

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.

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

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

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

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.

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.

During the last decade the number of characterized F420-dependent enzymes has significantly increased. Many of these deazaflavoproteins share a TIM-barrel fold and are structurally related to FMN-dependent luciferases and monooxygenases. In this work, we traced the origin and evolutionary history of the F420-dependent enzymes within the luciferase-like superfamily. By a thorough phylogenetic analysis we inferred that the F420-dependent enzymes emerged from a FMN-dependent common ancestor. Furthermore, the data show that during evolution, the family of deazaflavoproteins split into two well-defined groups of enzymes: the F420-dependent dehydrogenases and the F420-dependent reductases. By such event, the dehydrogenases specialized in generating the reduced deazaflavin cofactor, while the reductases employ the reduced F420 for catalysis. Particularly, we focused on investigating the dehydrogenase subfamily and demonstrated that this group diversified into three types of dehydrogenases: the already known F420-dependent glucose-6-phosphate dehydrogenases, the F420-dependent alcohol dehydrogenases, and the sugar-6-phosphate dehydrogenases that were identified in this study. By reconstructing and experimentally characterizing ancestral and extant representatives of F420-dependent dehydrogenases, their biochemical properties were investigated and compared. We propose an evolutionary path for the emergence and diversification of the TIM-barrel fold F420-dependent dehydrogenases subfamily. Fil: Mascotti, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina Fil: Kumar, Hemant. University of Groningen. Faculty of Mathematics and Natural Sciences; Países Bajos Fil: Nguyen, Quoc-Thai. Scuola Universitaria Superiore IUSS Pavia; Italia. University of Groningen. Faculty of Mathematics and Natural Sciences; Países Bajos. University of Medicine and Pharmacy; Vietnam Fil: Juri Ayub, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina Fil: Fraaije, Marco Wilhelmus. University of Groningen. Faculty of Mathematics and Natural Sciences; Países Bajos

The flavin-containing monooxygenases (FMOs) play an important role in drug metabolism but they also have a high potential in industrial biotransformations. Among the hitherto characterized FMOs, there was no thermostable representative, while such biocatalyst would be valuable for FMO-based applications. Through a targeted genome mining approach, we have identified a gene encoding for a putative FMO from Nitrincola lacisaponensis, an alkaliphilic extremophile bacterium. Herein, we report the biochemical and structural characterization of this newly discovered bacterial FMO (NiFMO). NiFMO can be expressed as active and soluble enzyme at high level in Escherichia coli (90-100 mg/L of culture). NiFMO is relatively thermostable (melting temperature (Tm) of 51 °C), displays high organic solvent tolerance, and accepts a broad range of substrates. The crystal structure of NiFMO was solved at 1.8 Å resolution, which allows future structure-based enzyme engineering. Altogether, NiFMO represents an interesting newly discovered enzyme with the appropriate features to develop into an industrially applied biocatalyst.

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.

One approach to bringing enzymes together for multienzyme biocatalysis is genetic fusion. This enables the production of multifunctional enzymes that can be used for whole‐cell biotransformations or for in vitro (cascade) reactions. In some cases and in some aspects, such as expression and conversions, the fused enzymes outperform a combination of the individual enzymes. In contrast, some enzyme fusions are greatly compromised in activity and/or expression. In this Minireview, we give an overview of studies on fusions between two or more enzymes that were used for biocatalytic applications, with a focus on oxidative enzymes. Typically, the enzymes are paired to facilitate cofactor recycling or cosubstrate supply. In addition, different linker designs are briefly discussed. Although enzyme fusion is a promising tool for some biocatalytic applications, future studies could benefit from integrating the findings of previous studies in order to improve reliability and effectiveness.

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.

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.

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

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

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

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.

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.