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Redox enzymes, mostly equipped with metal or organic cofactors, can vary their reactivity with oxygen by orders of magnitudes. Understanding how oxygen reactivity is controlled by the protein milieu remains an open issue with broad implications for mechanistic enzymology and enzyme design. Here, we address this problem by focusing on a widespread group of flavoenzymes that oxidize phenolic compounds derived from microbial lignin degradation, using either oxygen or a cytochrome c as electron acceptors. A comprehensive phylogenetic analysis revealed conserved amino acid motifs in their flavin-binding site. Using a combination of kinetics, mutagenesis, structural, and computational methods, we examined the role of these residues. Our results demonstrate that subtle and localized changes in the flavin environment can drastically impact on oxygen reactivity. These effects are afforded through the creation or blockade of pathways for oxygen diffusion. Substrate binding plays a crucial role by potentially obstructing oxygen access to the flavin, thus influencing the enzyme's reactivity. The switch between oxidase and dehydrogenase functionalities is thereby achieved through targeted, site-specific amino acid replacements that finely tune the microenvironment around the flavin. Our findings explain how very similar enzymes can exhibit distinct functional properties, operating as oxidases or dehydrogenases. They further provide valuable insights for the rational design and engineering of enzymes with tailored functions., Competing Interests: Conflict of Interest The authors declare that they have no competing interests with the content of this article.

Light-Oxygen-Voltage (LOV) domains are the protein-based light switches used in nature to trigger and regulate various processes. They allow light signals to be converted into metabolic signaling cascades. Various LOV-domain proteins have been characterized in the last few decades and have been used to develop light-sensitive tools in cell biology research. LOV-based applications exploit the light-driven regulation of effector elements to activate signaling pathways, activate genes, or locate proteins within cells. A relatively new application of an engineered small LOV-domain protein called miniSOG (mini singlet oxygen generator) is based on the light-induced formation of reactive oxygen species (ROS). The first miniSOG was engineered from a LOV domain from Arabidopsis thaliana. This engineered 14 kDa light-responsive flavin-containing protein can be exploited as protein tag for the light-triggered localized production of ROS. Such tunable ROS production by miniSOG or similarly redesigned LOV-domains can be of use in studies focused on subcellular phenomena but may also allow new light-fueled catalytic processes. This review provides an overview of the discovery of LOV domains and their development into tools for cell biology. It also highlights recent advancements in engineering LOV domains for various biotechnological applications and cell biology studies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial monoamine oxidases (MAOs) are FAD-dependent proteins catalyzing a relevant reaction for many industrial biocatalytic applications, ranging from production of enantiomerically pure building blocks for pharmaceutical synthesis to biosensors for monitoring food and beverage quality. The thermostable MAO enzyme from Thermoanaerobacterales bacterium (MAO Tb ) is about 36 % identical to both putrescine oxidase and human MAOs and can be efficiently produced in Escherichia coli. MAO Tb preferentially acts on n-alkyl monoamines but shows detectable activity also on polyamines and aromatic monoamines. The crystal structures of MAO Tb in complex with putrescine, benzylamine, spermidine and n-heptylamine at resolution ranging from 1.6 to 2.3 Å resolution revealed the binding mode of substrates to the enzyme. The MAO Tb active site is highly conserved in the inner part of the cavity in front of the flavin ring (re face), where the presence of two tyrosine residues creates the substrate amine binding site that is found also in human MAOs. Instead, more distantly from the flavin, the entrance of the catalytic site is much more open in MAO Tb and features a different arrangement of amino acids. Site-directed mutagenesis targeting residues Ala168, Thr199 and Val324 allowed the identification of key residues in ligand binding to alter substrate specificity. The A168D variant showed a higher activity on putrescine than wild-type, whereas by replacing either Thr199 or Val324 to Trp a marked enhancement in k cat /K M values was found on n-alkyl-monoamines and on aromatic amines., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Alditol oxidases are promising tools for the biocatalytic oxidation of glycerol to more valuable chemicals. By integrating in silico bioprospecting with cell-free protein synthesis and activity screening, an effective pipeline was developed to rapidly identify enzymes that are active on glycerol. Three thermostable alditol oxidases from Actinobacteria Bacterium, Streptomyces thermoviolaceus, and Thermostaphylospora chromogena active on glycerol were discovered. The characterization of these three flavoenzymes demonstrated their glycerol oxidation activities, preference for alkaline conditions, and excellent thermostabilities with melting temperatures higher than 75 °C. Structural elucidation of the alditol oxidase from Actinobacteria Bacterium highlighted a constellation of side chains that engage the substrate through several hydrogen bonds, a histidine residue covalently bound to the FAD prosthetic group, and a tunnel leading to the active site. Upon computational simulations of substrate binding, a double mutant targeting a residue pair at the tunnel entrance was created and found to display an improved thermal stability and catalytic efficiency for glycerol oxidation. The hereby described alditol oxidases form a valuable panel of oxidative biocatalysts that can perform regioselective oxidation of glycerol and other polyols. KEY POINTS: • Rapid pipeline designed to identify putative oxidases • Biochemical and structural characterization of alditol oxidases • Glycerol oxidation to more valuable derivatives., (© 2024. The Author(s).)

The vanillyl alcohol oxidase/p-cresol methylhydroxylase (VAO/PCMH) flavoprotein family comprises a broad spectrum of enzymes capable of catalyzing the oxidative bioconversions of various substrates. Among them, pinoresinol hydroxylase (PinH) from the 4-alkylphenol oxidizing subgroup initiates the oxidative degradation of (+)-pinoresinol, a lignan important for both lignin structure and plant defense. In this study, we present a detailed biochemical and structural characterization of PinH from Pseudomonas sp., with focus on its substrate specificity and product formation. PinH was expressed in E. coli and purified as FAD-containing, soluble protein. The flavoenzyme catalyzes the hydroxylation of both (+)-pinoresinol and eugenol. Structural analysis reveals its dimeric form, non-covalent flavin binding, and a large active site. AlphaFold models of the PinH-cytochrome complex demonstrate cytochrome's dual role in electron transfer and modulating PinH's conformation. A distinctive feature of PinH is a large cavity that hosts its multi-ring (+)-pinoresinol substrate. The capability of converting bulky lignans is particularly attractive for biotechnological applications aimed at producing high-value compounds from phenolic precursors. These insights expand our knowledge on the structure and mechanism of the VAO/PCMH flavoenzyme family members., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Previously, some bacteria were shown to harbour enzymes capable of catalysing the oxidative cleavage of the double bond of t-anethole and related compounds. The cofactor dependence of these enzymes remained enigmatic due to a lack of biochemical information. We report on catalytic and structural details of a representative of this group of oxidative enzymes: t-anethole oxygenase from Stenotrophomonas maltophilia (TAO Sm ). The bacterial enzyme could be recombinantly expressed and purified, enabling a detailed biochemical study that has settled the dispute on its cofactor dependence. We have established that TAO Sm contains a tightly bound b-type heme and merely depends on dioxygen for catalysis. It was found to accept t-anethole, isoeugenol and O-methyl isoeugenol as substrates, all being converted into the corresponding aromatic aldehydes without the need of any cofactor regeneration. The elucidated crystal structure of TAO Sm has revealed that it contains a unique active site architecture that is conserved for this distinct class of heme-containing bacterial oxygenases. Similar to other hemoproteins, TAO Sm has a histidine (His121) as proximal ligand. Yet, unique for TAOs, an arginine (Arg89) is located at the distal axial position. Site directed mutagenesis confirmed crucial roles for these heme-liganding residues and other residues that form the substrate binding pocket. In conclusion, the results reported here reveal a new class of bacterial heme-containing oxygenases that can be used for the cleavage of alkene double bonds, analogous to ozonolysis in organic chemistry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Peroxidases belong to a group of enzymes that are widely found in animals, plants and microorganisms. These enzymes are effective biocatalysts for a wide range of oxidations on various substrates. This work presents a biochemical and structural characterization of a novel heme-containing peroxidase from Cyanobacterium sp. TDX16, CyanoPOX. This cyanobacterial enzyme was successfully overexpressed in Escherichia coli as a soluble, heme-containing monomeric enzyme. Although CyanoPOX shares relatively low sequence identity (37 %) with bovine lactoperoxidase, it displays comparable biochemical properties. CyanoPOX is most stable and active in slightly acidic conditions (pH 6-6.5) and moderately thermostable (melting temperature around 48 °C). Several compounds that are typical substrates for mammalian lactoperoxidases were tested to establish the catalytic potential of CyanoPOX. Potassium iodide showed the highest catalytic efficiency (126 mM -1  s -1 ), while various aromatic compounds were also readily converted. Structural elucidation of CyanoPOX confirmed the presence of a non-covalently bound b-type heme cofactor that is situated in the central core of the protein. Except for a highly similar overall structure, CyanoPOX also has a conserved active site pocket when compared with mammalian lactoperoxidases. Due to its catalytic properties and high expression in a bacterial host, this newly discovered peroxidase shows promise for applications., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Pyranose oxidases are valuable biocatalysts, yet only a handful of bacterial pyranose oxidases are known. These bacterial enzymes exhibit noteworthy distinctions from their extensively characterized fungal counterparts, encompassing variations in substrate specificity and structural attributes. Herein a bacterial pyranose oxidase from Oscillatoria princeps (OPOx) was biochemically characterized in detail. In contrast to the fungal pyranose oxidases, OPOx could be well expressed in Escherichia coli as soluble, fully flavinylated, and active oxidase. It was found to be highly thermostable (melting temperature >90 °C) and showed activity on glucose, exhibiting an exceptionally low K M value (48 μM). Elucidation of its crystal structure revealed similarities with fungal pyranose oxidases, such as being a tetramer with a large central void leading to a narrow substrate access tunnel. In the active site, the FAD cofactor is covalently bound to a histidine. OPOx displays a relatively narrow pH optimum for activity with a sharp decline at relatively basic pH values which is accompanied by a drastic change in its flavin absorbance spectrum. The pH-dependent switch in flavin absorbance features and oxidase activity was shown to be fully reversible. It is hypothesized that a glutamic acid helps to stabilize the protonated form of the histidine that is tethered to the FAD. OPOx presents itself as a valuable biocatalyst as it is highly robust, well-expressed in E. coli, shows low K M values for monosaccharides, and has a peculiar pH-dependent "on-off switch"., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Prenylcysteine oxidases (PCYOXs) metabolize prenylated cysteines produced by protein degradation. They utilize oxygen as a co-substrate to produce free cysteine, an aldehyde, and hydrogen peroxide through the unusual oxidation of a thioether bond. In this study, we explore the evolution, structure, and mechanism of the two mammalian PCYOXs. A gene duplication event in jawed vertebrates originated in these two paralogs. Both enzymes are active on farnesyl- and geranylgeranylcysteine, but inactive on molecules with shorter prenyl groups. Kinetics experiments outline a mechanism where flavin reduction and re-oxidation occur rapidly without any detectable intermediates, with the overall reaction rate limited by product release. The experimentally determined three-dimensional structure of PCYOX1 reveals long and wide tunnels leading from the surface to the flavin. They allow the isoprene substrate to curl up within the protein and position its reactive cysteine group close to the flavin. A hydrophobic patch on the surface mediates membrane association, enabling direct substrate and product exchange with the lipid bilayer. Leveraging established knowledge of flavoenzyme inhibition, we designed sub-micromolar PCYOX inhibitors. Additionally, we discovered that PCYOXs bind and slowly degrade salisirab, an anti-RAS compound. This activity suggests potential and previously unknown roles of PCYOXs in drug metabolism., Competing Interests: Conflict of interest The authors declare that they have no competing interests with the content of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Berberine bridge enzyme-like oxidases are often involved in natural product biosynthesis and are seen as essential enzymes for the generation of intricate pharmacophores. These oxidases have the ability to transfer a hydride atom to the FAD cofactor, which enables complex substrate modifications and rearrangements including (intramolecular) cyclizations, carbon-carbon bond formations, and nucleophilic additions. Despite the diverse range of activities, the mechanistic details of these reactions often remain incompletely understood. In this Review, we delve into the complexity that BBE-like oxidases from bacteria, fungal, and plant origins exhibit by providing an overview of the shared catalytic features and emphasizing the different reactivities. We propose four generalized modes of action by which BBE-like oxidases enable the synthesis of natural products, ranging from the classic alcohol oxidation reactions to less common amine and amide oxidation reactions. Exploring the mechanisms utilized by nature to produce its vast array of natural products is a subject of considerable interest and can lead to the discovery of unique biochemical activities.

Nitroreductases (NRs) are NAD(P)H-dependent flavoenzymes that reduce nitro aromatic compounds to their corresponding arylamines via the nitroso and hydroxylamine intermediates. Because of their broad substrate scope and versatility, NRs have found application in multiple fields such as biocatalysis, bioremediation, cell-imaging and prodrug activation. However, only a limited number of members of the broad NR superfamily (> 24 000 sequences) have been experimentally characterized. Within this group of enzymes, only few are capable of amine synthesis, which is a fundamental chemical transformation for the pharmaceutical, agricultural, and textile industries. Herein, we provide a comprehensive description of a recently discovered NR from Bacillus tequilensis, named BtNR. This enzyme has previously been demonstrated to have the capability to fully convert nitro aromatic and heterocyclic compounds to their respective primary amines. In this study, we determined its biochemical, kinetic and structural properties, including its apparent melting temperature (T m ) of 59 °C, broad pH activity range (from pH 3 to 10) and a notably low redox potential (-236 ± 1 mV) in comparison to other well-known NRs. We also determined its steady-state and pre-steady-state kinetic parameters, which are consistent with other NRs. Additionally, we elucidated the crystal structure of BtNR, which resembles the well-characterized Escherichia coli oxygen-insensitive NAD(P)H nitroreductase (NfsB), and investigated the substrate binding in its active site through docking and molecular dynamics studies with four nitro aromatic substrates. Guided by these structural analyses, we probed the functional roles of active site residues by site-directed mutagenesis. Our findings provide valuable insights into the biochemical and structural properties of BtNR, as well as its potential applications in biotechnology., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Flavin-containing monooxygenase from Methylophaga sp. ( m FMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored m FMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis ( Ni FMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of m FMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations.

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii. Among a variety of aromatic substrates, RePT showed the highest substrate conversion for L-tryptophan and L-tyrosine (> 90%), yielding two mono-prenylated products in both cases. Nine phenolics from diverse phenolic subclasses were notably converted (> 10%), of which the stilbenes oxyresveratrol, piceatannol, pinostilbene, and resveratrol were the best acceptors (37-55% conversion). The position of prenylation was determined using NMR spectroscopy or annotated using MS 2 fragmentation patterns, demonstrating that RePT mainly catalyzed mono-O-prenylation on the hydroxylated aromatic substrates. On L-tryptophan, a non-hydroxylated substrate, it preferentially catalyzed C7 prenylation with reverse N1 prenylation as a secondary reaction. Moreover, RePT also possessed substrate-dependent organic solvent tolerance in the presence of 20% (v/v) methanol or DMSO, where a significant conversion (> 90%) was maintained. Our study demonstrates the potential of RePT as a biocatalyst for the production of bioactive prenylated aromatic amino acids, stilbenes, and various phenolic compounds. KEY POINTS: • RePT catalyzes prenylation of diverse aromatic substrates. • RePT enables O-prenylation of phenolics, especially stilbenes. • The novel RePT remains active in 20% methanol or DMSO., (© 2024. The Author(s).)

Enzymes are crucial for the emergence and sustenance of life on earth. How they became catalytically active during their evolution is still an open question. Two opposite explanations are plausible: acquiring a mechanism in a series of discrete steps or all at once in a single evolutionary event. Here, we use molecular phylogeny, ancestral sequence reconstruction, and biochemical characterization to follow the evolution of a specialized group of flavoprotein monooxygenases, the bacterial Baeyer-Villiger monooxygenases (BVMOs). These enzymes catalyze an intricate chemical reaction relying on three different elements: a reduced nicotinamide cofactor, dioxygen, and a substrate. Characterization of ancestral BVMOs shows that the catalytic mechanism evolved in a series of steps starting from a FAD-binding protein and further acquiring reactivity and specificity toward each of the elements participating in the reaction. Together, the results of our work portray how an intrinsically complex catalytic mechanism emerged during evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Arylamines are essential building blocks for the manufacture of valuable pharmaceuticals, pigments and dyes. However, their current industrial production involves the use of chemocatalytic procedures with a significant environmental impact. As a result, flavin-dependent nitroreductases (NRs) have received increasing attention as sustainable catalysts for more ecofriendly synthesis of arylamines. In this study, we assessed a novel NR from Bacillus tequilensis, named BtNR, for the synthesis of pharmaceutically relevant arylamines, including valuable synthons used in the manufacture of blockbuster drugs such as vismodegib, sonidegib, linezolid and sildenafil. After optimizing the enzymatic reaction conditions, high conversion of nitroaromatics to arylamines (up to 97 %) and good product yields (up to 56 %) were achieved. Our results indicate that BtNR has a broad substrate scope, including bulky nitro benzenes, nitro pyrazoles and nitro pyridines. Hence, BtNR is an interesting biocatalyst for the synthesis of pharmaceutically relevant amine-functionalized aromatics, providing an attractive alternative to traditional chemical synthesis methodologies., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Photosynthetic organisms, fungi, and animals comprise distinct pathways for vitamin C biosynthesis. Besides this diversity, the final biosynthetic step consistently involves an oxidation reaction carried out by the aldonolactone oxidoreductases. Here, we study the origin and evolution of the diversified activities and substrate preferences featured by these flavoenzymes using molecular phylogeny, kinetics, mutagenesis, and crystallographic experiments. We find clear evidence that they share a common ancestor. A flavin-interacting amino acid modulates the reactivity with the electron acceptors, including oxygen, and determines whether an enzyme functions as an oxidase or a dehydrogenase. We show that a few side chains in the catalytic cavity impart the reaction stereoselectivity. Ancestral sequence reconstruction outlines how these critical positions were affixed to specific amino acids along the evolution of the major eukaryotic clades. During Eukarya evolution, the aldonolactone oxidoreductases adapted to the varying metabolic demands while retaining their overarching vitamin C-generating function., (© 2024. The Author(s).)

Industrial application of the natural deazaflavin cofactor F 420 has high potential for the enzymatic synthesis of high value compounds. It can offer an additional range of chemistry to the use of well-explored redox cofactors such as FAD and their respective enzymes. Its limited access through organisms that are rather difficult to grow has urged research on the heterologous production of F 420 using more industrially relevant microorganisms such as Escherichia coli . In this study, we demonstrate the possibility of producing this cofactor in a robust and widely used industrial organism, Saccharomyces cerevisiae , by the heterologous expression of the F 420 pathway. Through careful selection of involved enzymes and some optimization, we achieved an F 420 yield of ∼1.3 μmol/L, which is comparable to the yield of natural F 420 producers. Furthermore, we showed the potential use of F 420 -producing S. cerevisiae for F 420 -dependent bioconversions by carrying out the whole-cell conversion of tetracycline. As the first demonstration of F 420 synthesis and use for bioconversion in a eukaryotic organism, this study contributes to the development of versatile bioconversion platforms.

Monoamine oxidases (MAOs) are pivotal regulators of neurotransmitters in mammals, while microbial MAOs have been shown to be valuable biocatalysts for enantioselective synthesis of pharmaceutical compounds or precursors thereof. To extend the knowledge of how MAOs function at the molecular level and in order to provide more biocatalytic tools, we set out to identify and study a robust bacterial variant: a MAO from the thermophile Thermoanaerobacterales bacterium (MAO Tb ). MAO Tb is highly thermostable with melting temperatures above 73 °C and is well expressed in Escherichia coli. Substrate screening revealed that the oxidase is most efficient with n-alkylamines with n-heptylamine being the best substrate. Presteady-state kinetic analysis shows that reduced MAO Tb rapidly reacts with molecular oxygen, confirming that it is a bona fide oxidase. The crystal structure of MAO Tb was resolved at 1.5 Å and showed an exceptionally high similarity with the two human MAOs, MAO A and MAO B. The active site of MAO Tb resembles mostly the architecture of human MAO A, including the cysteinyl protein-FAD linkage. Yet, the bacterial MAO lacks a C-terminal extension found in human MAOs, which explains why it is expressed and purified as a soluble protein, while the mammalian counterparts are anchored to the membrane through an α-helix. MAO Tb also displays a slightly different active site access tunnel, which may explain the specificity toward long aliphatic amines. Being an easy-to-express, thermostable enzyme, for which a high-resolution structure was elucidated, this bacterial MAO may develop into a valuable biocatalyst for synthetic chemistry or biosensing., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The chemical 5-hydroxymethylfurfural (HMF) can be derived from lignocellulose and is an interesting bio-based platform chemical as it has the potential to be transformed into numerous valuable building blocks such as the polymer-precursor 2,5-diformylfuran (DFF). To date, only a few oxidases acting on HMF are known and by sampling atypical species, we discovered a novel flavin-dependent oxidoreductase from the honeybee Apis mellifera (beeHMFO). The enzyme can perform the chemoselective oxidation of HMF to DFF but can also readily accept other aromatic alcohols as substrates. The function of the enzyme may well be the antimicrobial generation of hydrogen peroxide using HMF, which is very abundant in honey. The discovery of this insect-derived flavoprotein oxidase holds promising potential in the synthesis of renewable products and demonstrates that insects can be an interesting source of novel biocatalysts., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 μg·mL -1 PatE protein in the culture medium with a purity just under 90%., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Patulin synthase (PatE) from Penicillium expansum is a flavin-dependent enzyme that catalyses the last step in the biosynthesis of the mycotoxin patulin. This secondary metabolite is often present in fruit and fruit-derived products, causing postharvest losses. The patE gene was expressed in Aspergillus niger allowing purification and characterization of PatE. This confirmed that PatE is active not only on the proposed patulin precursor ascladiol but also on several aromatic alcohols including 5-hydroxymethylfurfural. By elucidating its crystal structure, details on its catalytic mechanism were revealed. Several aspects of the active site architecture are reminiscent of that of fungal aryl-alcohol oxidases. Yet, PatE is most efficient with ascladiol as substrate confirming its dedicated role in biosynthesis of patulin., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The drive for a circular bioeconomy has resulted in a great demand for renewable, biobased chemicals. We present a one-pot biocatalytic cascade reaction for the production of racemic syringaresinol, a lignan with applications as a nutraceutical and in polymer chemistry. The process consumes dihydrosinapyl alcohol, which can be produced renewably from the lignocellulosic material. To achieve this, a variant of eugenol oxidase was engineered for the oxidation of dihydrosinapyl alcohol into sinapyl alcohol with good conversion and chemoselectivity. The crystal structure of the engineered oxidase revealed the molecular basis of the influence of the mutations on the chemoselectivity of the oxidation of dihydrosinapyl alcohol. By using horseradish peroxidase, the subsequent oxidative dimerization of sinapyl alcohol into syringaresinol was achieved. Conditions for the one-pot, two-enzyme synthesis were optimized, and a high yield of syringaresinol was achieved by cascading the oxidase and peroxidase steps in a stepwise fashion. This study demonstrates the efficient production of syringaresinol from a compound that can be renewed by reductive catalytic fractionation of lignocellulose, providing a biocatalytic route for generating a valuable compound from lignin., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Ancestral sequence reconstruction (ASR) provides insight into the changes within a protein sequence across evolution. More specifically, it can illustrate how specific amino acid changes give rise to different phenotypes within a protein family. Over the last few decades it has established itself as a powerful technique for revealing molecular common denominators that govern enzyme function. Here, we describe the strength of ASR in unveiling catalytic mechanisms and emerging phenotypes for a range of different proteins, also highlighting biotechnological applications the methodology can provide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor. Yet, in 2016, a new type of thioredoxin reductase was discovered in Archaea which utilize instead a reduced deazaflavin cofactor (F 420 H 2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed that these two DFTRs are highly specific for F 420 H 2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that are dependent on NADPH (NTRs). A detailed structural analysis led to the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In recent years, studies have shown that a large number of bacteria secrete multi-flavinylated proteins. The exact roles and properties, of these extracellular flavoproteins that contain multiple covalently anchored FMN cofactors, are still largely unknown. Herein, we describe the biochemical and structural characterization of two multi-FMN-containing covalent flavoproteins, SaFMN3 from Streptomyces azureus and CbFMN4 from Clostridiaceae bacterium . Based on their primary structure, these proteins were predicted to contain three and four covalently tethered FMN cofactors, respectively. The genes encoding SaFMN3 and CbFMN4 were heterologously coexpressed with a flavin transferase (ApbE) in Escherichia coli , and could be purified by affinity chromatography in good yields. Both proteins were found to be soluble and to contain covalently bound FMN molecules. The SaFMN3 protein was studied in more detail and found to display a single redox potential (-184 mV) while harboring three covalently attached flavins. This is in line with the high sequence similarity when the domains of each flavoprotein are compared. The fully reduced form of SaFMN3 is able to use dioxygen as electron acceptor. Single domains from both proteins were expressed, purified and crystallized. The crystal structures were elucidated, which confirmed that the flavin cofactor is covalently attached to a threonine. Comparison of both crystal structures revealed a high similarity, even in the flavin binding pocket. Based on the crystal structure, mutants of the SaFMN3-D2 domain were designed to improve its fluorescence quantum yield by changing the microenvironment of the isoalloxazine moiety of the flavin cofactor. Residues that quench the flavin fluorescence were successfully identified. Our study reveals biochemical details of multi-FMN-containing proteins, contributing to a better understanding of their role in bacteria and providing leads to future utilization of these flavoprotein in biotechnology., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yapei Tong reports financial support was provided by CSC - Chinese scholarship Council., (© 2023 The Author(s).)

Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg -1 ) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg -1 ) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for ɣ-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg -1 ). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nitrosuccinate is a biosynthetic building block in many microbial pathways. The metabolite is produced by dedicated L-aspartate hydroxylases that use NADPH and molecular oxygen as co-substrates. Here, we investigate the mechanism underlying the unusual ability of these enzymes to perform successive rounds of oxidative modifications. The crystal structure of Streptomyces sp. V2 L-aspartate N-hydroxylase outlines a characteristic helical domain wedged between two dinucleotide-binding domains. Together with NADPH and FAD, a cluster of conserved arginine residues forms the catalytic core at the domain interface. Aspartate is found to bind in an entry chamber that is close to but not in direct contact with the flavin. It is recognized by an extensive H-bond network that explains the enzyme's strict substrate-selectivity. A mutant designed to create steric and electrostatic hindrance to substrate binding disables hydroxylation without perturbing the NADPH oxidase side-activity. Critically, the distance between the FAD and the substrate is far too long to afford N-hydroxylation by the C4a-hydroperoxyflavin intermediate whose formation is confirmed by our work. We conclude that the enzyme functions through a catch-and-release mechanism. L-aspartate slides into the catalytic center only when the hydroxylating apparatus is formed. It is then re-captured by the entry chamber where it waits for the next round of hydroxylation. By iterating these steps, the enzyme minimizes the leakage of incompletely oxygenated products and ensures that the reaction carries on until nitrosuccinate is formed. This unstable product can then be engaged by a successive biosynthetic enzyme or undergoes spontaneous decarboxylation to produce 3-nitropropionate, a mycotoxin., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Among the molecular mechanisms of adaptation in biology, enzyme functional diversification is indispensable. By allowing organisms to expand their catalytic repertoires and adopt fundamentally different chemistries, animals can harness or eliminate new-found substances and xenobiotics that they are exposed to in new environments. Here, we explore the flavin-containing monooxygenases (FMOs) that are essential for xenobiotic detoxification. Employing a paleobiochemistry approach in combination with enzymology techniques we disclose the set of historical substitutions responsible for the family's functional diversification in tetrapods. Remarkably, a few amino acid replacements differentiate an ancestral multi-tasking FMO into a more specialized monooxygenase by modulating the oxygenating flavin intermediate. Our findings substantiate an ongoing premise that enzymatic function hinges on a subset of residues that is not limited to the active site core., (© 2023. The Author(s).)

Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their high selectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recently discovered carbohydrate oxidase from the bacterium Ralstonia solanacearum , which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward N -acetyl-d-galactosamine and N -acetyl-d-glucosamine, the enzyme was named N -acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could be overexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analyses revealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystal structure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl and cysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodation of N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with a drastically modified substrate acceptance profile, tuned toward non-N-acetylated monosaccharides and disaccharides. Furthermore, the activity toward bulkier substrates such as the trisaccharide maltotriose was introduced by this mutation. Due to its advantage of being overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selective oxidation of monosaccharides and oligosaccharides.

Various 4-alkylphenols can be easily obtained through reductive catalytic fractionation of lignocellulosic biomass. Selective dehydrogenation of 4-n-propylguaiacol results in the formation of isoeugenol, a valuable flavor and fragrance molecule and versatile precursor compound. Here we present the engineering of a bacterial eugenol oxidase to catalyze this reaction. Five mutations, identified from computational predictions, are first introduced to render the enzyme more thermostable. Other mutations are then added and analyzed to enhance chemoselectivity and activity. Structural insight demonstrates that the slow catalytic activity of an otherwise promising enzyme variant is due the formation of a slowly-decaying covalent substrate-flavin cofactor adduct that can be remedied by targeted residue changes. The final engineered variant comprises eight mutations, is thermostable, displays good activity and acts as a highly chemoselective 4-n-propylguaiacol oxidase. We lastly use our engineered biocatalyst in an illustrative preparative reaction at gram-scale. Our findings show that a natural enzyme can be redesigned into a tailored biocatalyst capable of valorizing lignin-based monophenols., (© 2022. The Author(s).)

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif., Competing Interests: Conflict of interest The authors declare that there are no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Methods for facile site-selective modifications of proteins are in high demand. We have recently shown that a flavin transferase can be used for site-specific covalent attachment of a chromo- and fluorogenic flavin (FMN) to any targeted protein. Although this Flavin-tag method resulted in efficient labeling of proteins in vitro, labelling in E. coli cells resulted in partial flavin incorporation. It was also restricted in the type of installed label with only one type of flavin, FMN, being incorporated. Here, we report on an extension of the Flavin-tag method that addresses previous limitations. We demonstrate that co-expression of FAD synthetase improves the flavin incorporation efficiency, allowing complete flavin-labeling of a target protein in E. coli cells. Furthermore, we have found that various flavin derivatives and even a nicotinamide can be covalently attached to a target protein, rendering this method even more versatile and valuable., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

We describe the asymmetric synthesis of the most pleasant enantiomer of Jessemal fragrance. The key steps are (i) the one-pot reduction of an α-chloro-tetrasubstituted cyclohexenone to give the chlorohydrin, catalyzed by two stereoselective redox enzymes (an ene-reductase and an alcohol dehydrogenase); (ii) the regioselective epoxide ring-opening with organocuprate or organolithium nucleophiles. Density functional theory calculations together with the Curtin-Hammett principle allowed the rationalization of the regioselectivity.

Deazaflavin-dependent whole-cell conversions in well-studied and industrially relevant microorganisms such as Escherichia coli and Saccharomyces cerevisiae have high potential for the biocatalytic production of valuable compounds. The artificial deazaflavin FOP (FO-5'-phosphate) can functionally substitute the natural deazaflavin F 420 and can be synthesized in fewer steps, offering a solution to the limited availability of the latter due to its complex (bio)synthesis. Herein we set out to produce FOP in vivo as a scalable FOP production method and as a means for FOP-mediated whole-cell conversions. Heterologous expression of the riboflavin kinase from Schizosaccharomyces pombe enabled in vivo phosphorylation of FO, which was supplied by either organic synthesis ex vivo, or by a coexpressed FO synthase in vivo, producing FOP in E. coli as well as in S. cerevisiae . Through combined approaches of enzyme engineering as well as optimization of expression systems and growth media, we further improved the in vivo FOP production in both organisms. The improved FOP production yield in E. coli is comparable to the F 420 yield of native F 420 -producing organisms such as Mycobacterium smegmatis , but the former can be achieved in a significantly shorter time frame. Our E. coli expression system has an estimated production rate of 0.078 μmol L -1 h -1 and results in an intracellular FOP concentration of about 40 μM, which is high enough to support catalysis. In fact, we demonstrate the successful FOP-mediated whole-cell conversion of ketoisophorone using E. coli cells. In S. cerevisiae , in vivo FOP production by Sp RFK using supplied FO was improved through media optimization and enzyme engineering. Through structure-guided enzyme engineering, a Sp RFK variant with 7-fold increased catalytic efficiency compared to the wild type was discovered. By using this variant in optimized media conditions, FOP production yield in S. cerevisiae was 20-fold increased compared to the very low initial yield of 0.24 ± 0.04 nmol per g dry biomass. The results show that bacterial and eukaryotic hosts can be engineered to produce the functional deazaflavin cofactor mimic FOP.

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., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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 8α-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., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Carbohydrates are widely abundant molecules present in a variety of forms. For their biosynthesis and modification, nature has evolved a plethora of carbohydrate-acting enzymes. Many of these enzymes are of particular interest for biotechnological applications, where they can be used as biocatalysts or biosensors. Among the enzymes catalysing conversions of carbohydrates are the carbohydrate oxidases. These oxidative enzymes belong to different structural families and use different cofactors to perform the oxidation reaction of CH-OH bonds in carbohydrates. The variety of carbohydrate oxidases available in nature reflects their specificity towards different sugars and selectivity of the oxidation site. Thanks to their properties, carbohydrate oxidases have received a lot of attention in basic and applied research, such that nowadays their role in biotechnological processes is of paramount importance. In this review we provide an overview of the available knowledge concerning the known carbohydrate oxidases. The oxidases are first classified according to their structural features. After a description on their mechanism of action, substrate acceptance and characterisation, we report on the engineering of the different carbohydrate oxidases to enhance their employment in biocatalysis and biotechnology. In the last part of the review we highlight some practical applications for which such enzymes have been exploited., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The F 420 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 F 420 -dependent oxidoreductases. Five different deazaflavoenzyme Classes (I-V) are described according to their structural folds as follows: Class I encompassing the TIM-barrel F 420 -dependent enzymes; Class II including the Rossmann fold F 420 -dependent enzymes; Class III comprising the β-roll F 420 -dependent enzymes; Class IV which exclusively gathers the SH3 barrel F 420 -dependent enzymes and Class V including the three layer ββα sandwich F 420 -dependent enzymes. This classification provides a framework for the identification and biochemical characterization of novel deazaflavoenzymes., (© 2021 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

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 F 420 :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 F 420 H 2 was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with K m values in the millimolar range and k cat 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 k cat value of 4.2 s -1 toward 1-benzyl-3-acetylpyridine (BAP + ), which is similar to the k cat 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 ( Ts OYE). We were able to fully convert 10 mM ketoisophorone with BAP + within 16 h, using F 420 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., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

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.

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H 2 O 2 . 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 H 2 O 2 . While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised 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.

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 (PAMO RIAD -PTDH RIDD and PAMO RIAD/RIAD -PTDH RIDD ). 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.

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)