Flavoprotein Oxidases Research Articles

Biosynthetic Strategies of Berberine Bridge Enzyme-like Flavoprotein Oxidases toward Structural Diversification in Natural Product Biosynthesis.

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.

Biocatalytic Oxidative Amination of para‐Substituted Phenols

AbstractBiocatalytic preparation of chiral amines is a large and burgeoning field in organic chemistry. Many enzymes and routes have been published, including transaminases, imine reductases, reductive aminases, amine dehydrogenases and others. However, all these routes rely on some sacrificial substrate, in the form of either amine donor or cofactor regeneration substrate. Herein, we report the direct oxidative amination of p‐substituted phenols catalyzed by an evolved flavoprotein oxidase, with the consumption of only substrate and O2, and release of H2O2. The substrate scope of the reaction is studied, and is tolerant of a diverse panel including ammonia, primary and secondary amines, and amino acids. The reaction is later employed at preparative scale to generate aminated products in 50–80 % yield. This report establishes flavoprotein oxidase as a new and economical member of the chemist's toolkit for biocatalytic generation of chiral amines, acting as oxidative aminase.

Roles of flavoprotein oxidase and the exogenous heme- and quinone-dependent respiratory chain in lactic acid bacteria.

Lactic acid bacteria (LAB) are a type of bacteria that convert carbohydrates into lactate through fermentation metabolism. While LAB mainly acquire energy through this anaerobic process, they also have oxygen-consuming systems, one of which is flavoprotein oxidase and the other is exogenous heme- or heme- and quinone-dependent respiratory metabolism. Over the past two decades, research has contributed to the understanding of the roles of these oxidase machineries, confirming their suspected roles and uncovering novel functions. This review presents the roles of these oxidase machineries, which are anticipated to be critical for the future applications of LAB in industry and comprehending the virulence of pathogenic streptococci.

Efficient Oxidation of 5-Hydroxymethylfurfural Using a Flavoprotein Oxidase from the Honeybee Apis mellifera.

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.

Oxidative bicyclic ring system formation involving indole diterpene biosynthesis: Remarkable substrate tolerance of a prenyltransferase and flavoprotein oxidase

Oxidative modifications are key biosynthetic processes responsible for expanding the structural diversity of indole diterpenes (IDTs). In this study, we focused on the function of prenyltransferases and flavoprotein oxidases for the synthesis of nodulisporic acid-type and shearinine-type bicyclic rings found on the indole moiety. An in vitro enzymatic reaction of a prenyltransferase JanD, which mediates diprenylation at the C5- and C6-positions, revealed that JanD recognizes a C13‑hydroxy group on the diterpene moiety. Subsequent enzymatic reactions with flavoprotein oxidases, JanO, and NodO, indicated that JanO can accommodate penta/hexacyclic IDTs, while NodO exhibits strict substrate specificity toward pentacyclic IDTs. The broad substrate tolerance of JanD and JanO provides opportunities for the synthesis of unnatural IDTs based on artificial reconstitution in a heterologous host.

Insights into Flavoenzymes: Structure, classification and potential biotechnological applications

Flavoenzymes are the diverse enzymes that use the flavin cofactors for their catalysis. They are widespread from bacteria to humans and have various biological role. This review provides a comprehensive overview about the flavoenzymes, classification, catalysis mechanism and their applications. The transfer of electrons between the flavin molecule which possess basically the isoalloxazine chromophore comprises of 7,8-dimethylbenzene moiety and the hydrophilic pyrimidine ring combine to form the amphipathic molecule, and the substrate which forms the valuable products by undergone chemical transformation which also provides basis for their classification. Here mainly, dehydrogenases, flavoprotein oxidases, monooxygenases and reductases classes are explained with examples along with their many physiological roles, showcasing their involvement in essential cellular functions. However, some additional redox centers flavoproteins, metal-containg, flavocytochromes and disulphide oxidoreductases are also mentioned here. Beside that they have many other tremendous applications including in disease diagnosis, treatments, novel drugs and therapeutic approaches for various diseases, biosensors, industrial, various biotechnological applications and some recent achieved advancements are discussed in detail. This study aims to advance knowledge of these unique enzymes by providing an extensive analysis of the classification, processes, and uses of flavoenzymes. Further study of flavoenzyme catalysis is required to unveil exciting avenues for future study and invention.

Photochemical and Molecular Dynamics Studies of Halide Binding in Flavoenzyme Glucose Oxidase.

Glucose oxidase (GOX), a characteristic flavoprotein oxidase with widespread industrial applications, binds fluoride (F- ) and chloride (Cl- ). We investigated binding properties of halide inhibitors of GOX through time-resolved spectral characterization of flavin-related photochemical processes and molecular dynamic simulations. Cl- and F- bind differently to the protein active site and have substantial but opposite effects on the population and decay of the flavin excited state. Cl- binds closer to the flavin, whose excited-state decays in <100 fs due to anion-π interactions. Such interactions appear absent in F- binding, which, however, significantly increases the active-site rigidity leading to more homogeneous, picosecond fluorescence decay kinetics. These findings are discussed in relation to the mechanism of halide inhibition of GOX by occupying the accommodation site of catalytic intermediates and increasing the active-site rigidity.

Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase.

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.

Ultrafast photooxidation of protein-bound anionic flavin radicals

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Systematic Assessment of Uncoupling in Flavoprotein Oxidases and Monooxygenases

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.

Mechanistic Study of Oxidoreductase AprQ Involved in Biosynthesis of Aminoglycoside Antibiotic Apramycin

Main observation and conclusionThe aminoglycoside antibiotic apramycin contains a unique bicyclic octose moiety, and biosynthesis of this moiety involves an oxidoreductase AprQ. Unlike other known “Q” series proteins involved in aminoglycosides biosynthesis, AprQ does not work with an aminotransferase partner, and performs a four‐electron oxidation that converts a CH2OH moiety to a carboxylate group. In this study, we report mechanistic investigation of AprQ. We showed AprQ contains a flavin mononucleotide (FMN) cofactor, which is different from other known Q series enzymes that contain a flavin adenine dinucleotide (FAD) cofactor. A series of biochemical assays showed that AprQ is not a monooxygenase but a flavoprotein oxidase. Although molecular O2 is strictly required for reaction turnover, four‐electron oxidation can be achieved in the absence of O2 in single turnover condition. These findings establish the detailed catalytic mechanism of AprQ and expand the growing family of flavoprotein oxidases, an increasingly important class of biocatalysts.

Enantioselective oxidation of secondary alcohols by the flavoprotein alcohol oxidase from Phanerochaete chrysosporium

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.

Enzymatic spiroketal formation via oxidative rearrangement of pentangular polyketides

The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps. This is exemplified by the generation of the bisbenzannulated [5,6]-spiroketal pharmacophore in the bacterial rubromycin family of aromatic polyketides, which exhibit a wide array of bioactivities such as the inhibition of HIV reverse transcriptase or DNA helicase. Here we elucidate the complex flavoenzyme-driven formation of the rubromycin pharmacophore that is markedly distinct from conventional (bio)synthetic strategies for spiroketal formation. Accordingly, a polycyclic aromatic precursor undergoes extensive enzymatic oxidative rearrangement catalyzed by two flavoprotein monooxygenases and a flavoprotein oxidase that ultimately results in a drastic distortion of the carbon skeleton. The one-pot in vitro reconstitution of the key enzymatic steps as well as the comprehensive characterization of reactive intermediates allow to unravel the intricate underlying reactions, during which four carbon-carbon bonds are broken and two CO2 become eliminated. This work provides detailed insight into perplexing redox tailoring enzymology that sets the stage for the (chemo)enzymatic production and bioengineering of bioactive spiroketal-containing polyketides.

Associations between renalase concentration and the occurrence of selected diseases.

Renalase is a recently identified flavoprotein oxidase, secreted mainly by the kidneys, which takes part in the degradation of catecholamines. The catecholamine inactivating effect results in the modulation of the sympathetic system tension and, consequently, in a decrease of blood pressure, myocardial contractility, heart rate, and vascular tone. Besides its enzymatic capacity, renalase shows cytoprotective properties by activating mitogen-activated protein kinase (MAPK) pathway. Several single nucleotide polymorphisms (SNPs) of the renalase gene have been identified, of which the most widely studied in relation to the development of selected diseases are rs2296545, rs10887800, and rs2576178. Numerous publications prove the contribution of renalase to the occurrence of cardiovascular diseases, kidney diseases, ischaemic stroke, diabetes type 1 and 2, as well as female infertility and schizophrenia. Further extended research into the various mechanisms of renalase activity may result in the use of this oxidase or its analogues as a therapeutic and/or diagnostic tool.

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase.

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.

The multipurpose family of flavoprotein oxidases.

This chapter represents a journey through flavoprotein oxidases. The purpose is to excite the reader curiosity regarding this class of enzymes by showing their diverse applications. We start with a brief overview on oxidases to then introduce flavoprotein oxidases and elaborate on the flavin cofactors, their redox and spectroscopic characteristics, and their role in the catalytic mechanism. The six major flavoprotein oxidase families will be described, giving examples of their importance in biology and their biotechnological uses. Specific attention will be given to a few selected flavoprotein oxidases that are not extensively discussed in other chapters of this book. Glucose oxidase, cholesterol oxidase, 5-(hydroxymethyl)furfural (HMF) oxidase and methanol oxidase are four examples of oxidases belonging to the GMC-like flavoprotein oxidase family and that have been shown to be valuable biocatalysts. Their structural and mechanistic features and recent enzyme engineering will be discussed in details. Finally we give a look at the current trend in research and conclude with a future outlook.

The Mechanism of Sugar C-H Bond Oxidation by a Flavoprotein Oxidase Occurs by a Hydride Transfer Before Proton Abstraction.

Understanding the reaction mechanism underlying the functionalization of C-H bonds by an enzymatic process is one of the most challenging issues in catalysis. Here, combined approaches using density functional theory (DFT) analysis and transient kinetics were employed to investigate the reaction mechanism of C-H bond oxidation in d-glucose, catalyzed by the enzyme pyranose 2-oxidase (P2O). Unlike the mechanisms that have been conventionally proposed, our findings show that the first step of the C-H bond oxidation reaction is a hydride transfer from the C2 position of d-glucose to N5 of the flavin to generate a protonated ketone sugar intermediate. The proton is then transferred from the protonated ketone intermediate to a conserved residue, His548. The results show for the first time how specific interactions around the sugar binding site promote the hydride transfer and formation of the protonated ketone intermediate. The DFT results are also consistent with experimental results including the enthalpy of activation obtained from Eyring plots, as well as the results of kinetic isotope effect and site-directed mutagenesis studies. The mechanistic model obtained from this work may also be relevant to other reactions of various flavoenzyme oxidases that are generally used as biocatalysts in biotechnology applications.

Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design

BackgroundHMF oxidase (HMFO) from Methylovorus sp. is a recently characterized flavoprotein oxidase. HMFO is a remarkable enzyme as it is able to oxidize 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA): a catalytic cascade of three oxidation steps. Because HMF can be formed from fructose or other sugars and FDCA is a polymer building block, this enzyme has gained interest as an industrially relevant biocatalyst.ResultsTo increase the robustness of HMFO, a requirement for biotechnological applications, we decided to enhance its thermostability using the recently developed FRESCO method: a computational approach to identify thermostabilizing mutations in a protein structure. To make this approach even more effective, we now developed a new and facile gene shuffling approach to rapidly combine stabilizing mutations in a one-pot reaction. This allowed the identification of the optimal combination of seven beneficial mutations. The created thermostable HMFO mutant was further studied as a biocatalyst for the production of FDCA from HMF and was shown to perform significantly better than the original HMFO.ConclusionsThe described new gene shuffling approach quickly discriminates stable and active multi-site variants. This makes it a very useful addition to FRESCO. The resulting thermostable HMFO variant tolerates the presence of cosolvents and also remained thermotolerant after introduction of additional mutations aimed at improving the catalytic activity. Due to its stability and catalytic efficiency, the final HMFO variant appears to be a promising candidate for industrial scale production of FDCA from HMF.

Kinetic Resolution of sec-Thiols by Enantioselective Oxidation with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase.

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.

Kinetic Resolution of sec‐Thiols by Enantioselective Oxidation with Rationally Engineered 5‐(Hydroxymethyl)furfural Oxidase

AbstractVarious 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.