The glucose-methanol-choline (GMC) superfamily is a large and functionally diverse family of oxidoreductases that share a common structural fold. Fungal members of this superfamily that are characterised and relevant for lignocellulose degradation include aryl-alcohol oxidoreductase, alcohol oxidase, cellobiose dehydrogenase, glucose oxidase, glucose dehydrogenase, pyranose dehydrogenase, and pyranose oxidase, which together form family AA3 of the auxiliary activities in the CAZy database of carbohydrate-active enzymes. Overall, little is known about the extant sequence space of these GMC oxidoreductases and their phylogenetic relations. Although some individual forms are well characterised, it is still unclear how they compare in respect of the complete enzyme class and, therefore, also how generalizable are their characteristics.

The GMC oxidoreductases comprise a large family of diverse FAD enzymes that share a homologous backbone. The relationship and origin of the GMC oxidoreductase genes, however, was unknown. Recent sequencing of entire genomes has allowed for the evolutionary analysis of the GMC oxidoreductase family.

Curdlan (β-1,3-glucan), as a biodegradable polymer, is still an underestimated but potentially attractive matrix for the production of dressing materials. However, due to its lack of susceptibility to functionalization, its use is limited. The proposed curdlan modification, using a functional polycatecholamine layer, enables the immobilization of selected oxidoreductases (laccase and peroxidase) on curdlan hydrogel. The following significant changes of biological and mechanical properties of polycatecholamines + oxidoreductases-modified matrices were observed: reduced response of human monocytes in contact with the hydrogels, modulated reaction of human blood, in terms of hemolysis and clot formation, and changed mechanical properties. The lack of toxicity towards human fibroblasts and the suppression of cytokines released by human monocytes in comparison to pristine curdlan hydrogel, seems to make the application of such modifications attractive for biomedical purposes. The obtained results could also be useful for construction of a wide range of biomaterials based on other polymer hydrogels.

Biological properties of protein molecules depend on their interaction with other molecules, and enzymes are no exception. Enzyme activities are controlled by their interaction with other molecules in living cells. Enzyme activation and their catalytic properties in the presence of different types of polymers have been studied in vitro, although these studies are restricted to only a few enzymes. In this study, we show that addition of poly-l-lysine (PLL) can increase the enzymatic activity of multiple oxidoreductases through formation of enzyme assemblies. Oxidoreductases with an overall negative charge, such as l-lactate oxidase, d-lactate dehydrogenase, pyruvate oxidase, and acetaldehyde dehydrogenase, each formed assemblies with the positively charged PLL via electrostatic interactions. The enzyme activities of these oxidoreductases in the enzyme assemblies were several-folds higher than those of the enzyme in their natural dispersed state. In the presence of PLL, the turnover number (kcat) improved for all enzymes, whereas the decrease in Michaelis constant (KM) was enzyme dependent. This type of enzyme function regulation through the formation of assemblies via simple addition of polymers has potential for diverse applications, including various industrial and research purposes.

Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundation for the development of therapeutic strategies.

Oxidoreductases are major enzymes of xenobiotic metabolism. Consequently, they are essential in the chemoprotection of the human body. Many xenobiotic metabolism enzymes have been shown to be involved in chemosensory tissue protection. Among them, some were additionally shown to be involved in chemosensory perception, acting in signal termination as well as in the generation of metabolites that change the activation pattern of chemosensory receptors. Oxidoreductases, especially aldehyde dehydrogenases and aldo-keto reductases, are the first barrier against aldehyde compounds, which include numerous odorants. Using a mass spectrometry approach, we characterized the most highly expressed members of these families in the human nasal mucus sampled in the olfactory vicinity. Their expression was also demonstrated using immunohistochemistry in human epitheliums sampled in the olfactory vicinity. Recombinant enzymes corresponding to three highly expressed human oxidoreductases (ALDH1A1, ALDH3A1, AKR1B10) were used to demonstrate the high enzymatic activity of these enzymes toward aldehyde odorants. The structure‒function relationship set based on the enzymatic parameters characterization of a series of aldehyde odorant compounds was supported by the X-ray structure resolution of human ALDH3A1 in complex with octanal.

A gram-positive bacterium, denominated CFA-06, was isolated from Brazilian petroleum in the Campos Basin and is responsible for the degradation of aromatic compounds and petroleum aromatic fractions. The CFA-06 strain was identified as Bacillus safensis using the 16S rRNA and gyrase B sequence. Enzymatic assays revealed the presence of two oxidoreductases: a catalase and a new oxidoreductase. The oxidoreductases were enzymatically digested and analyzed via ESI-LTQ-Orbitrap mass spectrometry. The mass data revealed a novel oxidoreductase (named BsPMO) containing 224 amino acids and 89% homology with a hypothetic protein from B. safensis (CFA-06) and a catalase (named BsCat) with 491 amino acids and 60% similarity with the catalase from Bacillus pumilus (SAFR-032). The new protein BsPMO contains iron atom(s) and shows catalytic activity toward a monooxygenase fluorogenic probe in the presence of cofactors (NADH, NADPH and NAD). This study enhances our knowledge of the biodegradation process of petroleum by B. safensis.

Irc24p is a benzil oxidoreductase encoded on chromosome IX of Saccharomyces cerevisiae . We identified a putative paralog, Nre1p, encoded 284 bp downstream. Both proteins are small, cytoplasmic, and are 52% identical (70% similar). PANTHER and PFAM analysis of the amino acid sequences and rigid pairwise structure alignment predicted a conserved active site and Rossmann folds in both, implicating NADH or NADPH as likely cofactors. We purified hexahistidine-tagged Irc24p and Nre1p. Both proteins catalyze the reduction of the diketone benzil with similar kinetics and a preference for NADPH. This is the first demonstration of in vitro function for Nre1p.

F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases.

The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. The lumen of the mammalian ER contains >20 members of the protein disulfide isomerase (PDI) superfamily, which ensure formation of the correct set of intramolecular and intermolecular disulfide bonds as crucial, rate-limiting reactions of the protein folding process. Components of the PDI superfamily may also facilitate dislocation of misfolded polypeptides across the ER membrane for ER-associated degradation (ERAD). The reasons for the high redundancy of PDI family members and the substrate features required for preferential engagement of one or the other are poorly understood. Here we show that TMX1, one of the few transmembrane members of the family, forms functional complexes with the ER lectin calnexin and preferentially intervenes during maturation of cysteine-containing, membrane-associated proteins while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane. As such, TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.

As the first three-dimensional structure of the two-subunit type 2-oxoacid:ferredoxin oxidoreductases (OFOR) from archaea, we solved the crystal structures of STK_23000/STK_22980 (StOFOR1) and STK_24350/STK_24330 (StOFOR2) from Sulfolobus tokodaii. They showed similar overall structures, consisting of two a- and b-subunit heterodimers containing thiamin pyrophosphate (TPP) cofactor and [4Fe-4S] cluster, but lack an intramolecular ferredoxin domain. Unlike other OFORs, StOFORs can utilize both pyruvate and 2-oxoglutarate, playing a key role in the central metabolism. In the structure of StOFOR2 in unreacted pyruvate complex form, carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the a-subunit, which are conserved in pyruvate:ferredoxin oxidoreductase from Desulfovbrio africanus (DaPFOR). In the structure of StOFOR1 co-crystallized with 2-oxobutyrate, electron density corresponding to a 1-hydroxypropyl group (post-decarboxylation state) was observed at the thiazole ring of TPP. The binding pockets of the StOFORs surrounding the methyl or propyl group of the ligands are wider than that of DaPFOR. Mutational analyses indicated that several residues were responsible for the broad 2-oxoacid specificity of StOFORs. We also constructed a possible complex structural model by placing a Zn(2+)-containing dicluster ferredoxin of S. tokodaii into the large pocket of StOFOR2, providing insight into the electron transfer between the two redox proteins.

Chlorophyll biosynthesis enables autotrophic development of developing seedlings. Upon light exposure, the chlorophyll precursor protochlorophyllide produces reactive oxygen species (ROS). Developing seedlings acquire photosynthetic competence through the action of protochlorophyllide oxidoreductases (PORs) that convert protochlorophyllide to chlorophyllide, reducing ROS production that would otherwise induce cellular damage and chlorophyll bleaching. Here, we show that FCA mediates the thermostabilization of PORs to trigger the conversion of protochlorophyllide to chlorophyllide in developing seedlings. FCA also facilitates the thermal induction of POR genes through histone acetylation that promotes the accessibility of RNA polymerases to the gene promoters. The combined action of FCA maintains PORs at warm temperatures, shifting the chlorophyll-ROS balance toward autotrophic development. We propose that the FCA-mediated thermal adaptation of autotrophic development allows developing seedlings to cope with the heat-absorbing soil surface layer under natural conditions. The thermal adaptive mechanism would provide a potential basis for studying crop performance at warm temperatures.

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a redox-dependent enzymatic activity with glutathione or not. Investigation of enzymatic activity traditionally involved time-consuming in vitro characterization of recombinant proteins, limiting the capacity to study novel mechanisms and structure-function relationships. To accelerate our investigation of glutathione-dependent oxidoreductases, we have developed a high-throughput and semi-quantitative assay in yeast. We combined overexpression of the glutathione transporter OPT1 with genetic fusion constructs between glutathione-dependent oxidoreductases and redox-sensitive green fluorescent protein 2 (roGFP2) to allow the rapid characterization of enzymatic activity with physiological substrates. We show that the kinetics of roGFP2 oxidation by glutathione disulfide correlate well with the in vitro-determined activity of the genetically fused glutaredoxins or mutants thereof. Our assay thus allows direct screening of glutaredoxin activity and rapid investigation of structure-function relationships. We also demonstrate that our assay can be used to monitor roGFP2 oxidation by S-nitrosoglutathione (GSNO). We show that glutaredoxins efficiently catalyze oxidation of roGFP2 by GSNO in both live yeast cells and in vitro. In summary, we have established a novel assay for activity screening and characterization of glutathione-dependent oxidoreductases.

In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium.

The CAZy auxiliary activity family 3 (AA3) comprises FAD-dependent enzymes belonging to the superfamily of glucose-methanol-choline (GMC) oxidoreductases. Glucose oxidase (GOx; EC 1.1.3.4) and glucose dehydrogenase (GDH; EC 1.1.5.9) are part of subfamily AA3_2 and catalyze the oxidation of β-D-glucose at its anomeric carbon to D-glucono-1,5-lactone. Recent phylogenetic analysis showed that AA3_2 glucose oxidoreductases can be grouped into four major clades, GOx I and GDH I-III, and in minor clades such as GOx II or distinct subclades. This wide sequence space of AA3_2 glucose oxidoreductases has, however, not been studied in detail, with mainly members of GOx I and GDH I studied biochemically or structurally. Here, we report the biochemical characterization of four fungal glucose oxidoreductases from distinct, hitherto unexplored clades or subclades. The enzyme from Aureobasidium subglaciale, belonging to the minor GOx II clade, showed a typical preference for oxygen and glucose, confirming the correct annotation of this clade. The other three enzymes exhibited strict dehydrogenase activity with different substrate specificities. GDH II from Trichoderma virens showed an almost six-fold higher catalytic efficiency for maltose compared to glucose. The preferred substrate for the two GDH III enzymes from Rhizoctonia solani and Ustilago maydis was gentiobiose, a β(1→6) disaccharide, as judged from the catalytic efficiency. Overall, the newly studied AA3_2 glucose oxidoreductases showed a much broader substrate spectrum than the archetypal GOx from Aspergillus niger, which belongs to clade GOx I.

Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability.

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 three layer ββα sandwich F420 -dependent enzymes. This classification provides a framework for the identification and biochemical characterization of novel deazaflavoenzymes.

Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α-associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a' domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells.

The replication machinery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely associated with the endoplasmic reticulum (ER) in host cells. Activation of the unfolded protein response (UPR) is a strategy hijacked by coronavirus to facilitate its replication and suppress host innate immunity. Here, we have found that SARS-CoV-2 ORF8 protein accumulates in the ER and escapes the degradation system by forming mixed disulfide complexes with ER oxidoreductases. ORF8 induces the activation of three UPR pathways through targeting key UPR components, remodels ER morphology and accelerates protein trafficking. Moreover, small molecule reducing agents release ORF8 from the mixed disulfide complexes and facilitate its degradation, therefore mitigate ER stress. Our study reveals a unique mechanism by which SARS-CoV-2 ORF8 escapes degradation by host cells and regulates ER reshaping. Targeting ORF8-involved mixed disulfide complexes could be a new strategy to alleviate SARS-CoV-2 induced ER stress and related diseases.