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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.

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.

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.

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

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.

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

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

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.

A collection of fusion biocatalysts has been generated that can be used for self-sufficient oxygenations or ketone reductions. These biocatalysts were created by fusing a Baeyer-Villiger monooxygenase (cyclohexanone monooxygenase from Thermocrispum municipale: TmCHMO) or an alcohol dehydrogenase (alcohol dehydrogenase from Lactobacillus brevis: LbADH) with three different cofactor regeneration enzymes (formate dehydrogenase from Burkholderia stabilis: BsFDH; glucose dehydrogenase from Sulfolobus tokodaii: StGDH, and phosphite dehydrogenase from Pseudomonas stutzeri: PsPTDH). Their tolerance against various organic solvents, including a deep eutectic solvent, and their activity and selectivity with a variety of substrates have been studied. Excellent conversions and enantioselectivities were obtained, demonstrating that these engineered fusion enzymes can be used as biocatalysts for the synthesis of (chiral) valuable compounds. (C) 2019 The Authors. Published by Elsevier Ltd.

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

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.

Dye decolorizing peroxidases (DyPs) are novel haem-containing peroxidases, which are structurally unrelated to classical peroxidases. They lack the highly conserved distal histidine that acts as an acidbase catalyst in the catalytic reaction of classical peroxidases, which implies distinct mechanistic properties. Despite the remarkable catalytic properties and recognized potential for biotechnology applications, the knowledge of DyP's structural features in solution, which govern the reactivity and catalysis, is lagging behind. Resonance Raman (RR) spectroscopy can reveal fine details of the active site structure in hemoproteins, reporting on the oxidation and spin state and coordination of the haem cofactor. We provide an overview of the haem binding pocket architecture of the enzymes from A, B and C DyP subfamilies, in the light of those established for classical peroxidases and search for subfamily specific features among DyPs. RR demonstrates that multiple spin populations typically co-exist in DyPs, like in the case of classical peroxidases. The haem spin/coordination state is strongly pH dependent and correlates well with the respective catalytic properties of DyPs. Unlike in the case of classical peroxidases, a surprisingly high abundance of catalytically incompetent low spin population is observed in several DyPs, and tentatively related to the alternative physiological function of these enzymes. The molecular details of active sites of DyPs, elucidated by RR spectroscopy, can furthermore guide approaches for biotechnological exploitation of these promising biocatalysts.

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.

Biochemical and biophysical research communications 529(3), 548 - 553 (2020). doi:10.1016/j.bbrc.2020.06.059, Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5–9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity., Published by Academic Press, Orlando, Fla.

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

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

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

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.

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.