Selenocysteine-dependent Enzymes: Structure, Function and Selenium-derived Mechanism.

Multi-subunit formate dehydrogenases (FDHs), which catalyze the interconversion of formate and carbon dioxide (CO2), have drawn increasing attention for mitigating climate change and advancing environmental protection owing to their advantages of oxygen tolerance and easy heterogenous expression. However, differently sourced multi-subunit FDHs exhibit distinct catalytic biases, and the reasons remain unclear. On the basis of the exceptional observation of Rhodobacter aestuarii FDH favoring CO2 reduction, this study unveiled an oxidation inhibition effect in exclusively NADH/NAD+-involved catalysis via kinetics analysis in terms of different redox couples. Substrate truncation positioned Fdhβ as the predominant subunit. Further studies based on structural and electrochemical insights interpreted that the slow desorption of NADH is the underlying determinant for the apparent catalytic bias. Knowledge-based rational design helped obtain a beneficial variant, RaFDH β E260Y, with a 10-fold increased catalytic activity in CO2 reduction, highlighting its potential for CO2 biotransformation and applications in low-carbon biomanufacturing. Eventually, bioinformatic analysis suggested that the diaphorase-like subunits and the catalysis regulation mechanism may widely exist in living organisms for modulating the redox balance of oxidoreductases, providing new insights into metabolism and catabolism.

Direct electron transfer-type NADH regeneration flow reactor with recombinant diaphorase subunit of formate dehydrogenase 1.

We report experiments that probe the role of the NAD+-driven protein conformational change in the formate dehydrogenase(FDH)-catalyzed hydride transfer. The binding interactions betweenFDH and the ADP fragment of NAD+ provide an 11.9 kcal/molstabilization of the transition state for FDH-catalyzed hydride transferfrom formate to NAD+ and a 7.9 kcal/mol stabilization ofthe complex between FDH and the putative transition state analogueazide anion. The binding interactions between FDH and the AMP cofactorpiece likewise provide a 5.6 kcal/mol stabilization of the transitionstate for FDH-catalyzed hydride transfer from formate to nicotinamideriboside (NR) and a 1.4 kcal/mol stabilization of the complex betweenFDH and the azide anion. The results provide support for the conclusionthat binding of NAD+ or the AMP cofactor fragment to FDHdrives a change in protein conformation from a flexible open conformationto the tight closed conformation that locks the active site side chainsinto positions that provide optimal stabilization of both the hydridetransfer transition state and the azide anion mimic for this transitionstate.

This study reports scalable bioelectrodes for sustainable CO2-to-formate conversion that integrate on-electrode cofactor regeneration with enzyme immobilization. Carbon felt (CF) supports were coated with copper (Cu) or tin oxide (SnO2) nanoparticles, allowing for reproducible and straightforward fabrication. Electrochemical characterization revealed that Cu-modified electrodes (CF-NpCu) outperformed SnO2-modified ones (CF-NpSnO2) in NADH regeneration, achieving nearly double the faradaic efficiency (FE) toward formate and conversion yield. Coupling CF-NpCu electrodes with affinity-immobilized formate dehydrogenase (FDH) produced 4.4 mM formate after 5 h, a threefold increase compared to the free enzyme system. Although the free enzyme displayed higher intrinsic kinetics, immobilization positioned FDH proximal to the electrode, mitigating diffusional limitations, accelerating NADH turnover, and improving stability. The integrated system achieved a productivity of 43 µmol h-1 cm-2 and demonstrated reusability, highlighting its practical applicability. Despite moderate efficiency losses due to side reactions such as hydrogen evolution, this work establishes a scalable bioelectrode platform that effectively combines cofactor regeneration with enzymatic CO2 reduction, providing a promising route toward sustainable and industrially relevant electroenzymatic processes.

This paper describes the electroenzymatic reduction of CO2to formate catalyzed by formate dehydrogenase fromCandida boidinii(CbFDH) immobilized on carbon nanotube (CNT)-modified gold electrodes. Cyclic voltammetry indicates thatCbFDH could catalyze CO2reduction to formate without protonated nicotinamide adenine dinucleotide (NADH) as a cofactor, exhibiting diffusion-controlled, quasi-reversible kinetics on both multi-walled CNT and single-walled CNT substrates. Surface-enhanced infrared absorption spectra indicate thatCbFDH adopts a near-parallel orientation on the CNT-modified gold surface, positioning its active site for the direct electron transfer between CO2and the conductive carbon support. The IR spectra reveal an increase in the formate band's intensity in the potential region from -0.3 V to -0.6 V vs Ag/AgCl, confirming efficient CO2reduction. Below -0.6 V vs Ag/AgCl, the hydrogen evolution reaction competitively suppresses formate yield. This study demonstrates that CNTs serve as an effective support for enzyme immobilization and confirms that CO2could be directly reduced to formate at the CNT-modified electrode without a cofactor at potentials close to the equilibrium potential (minimum of overpotential). This represents a novel and unexpected finding.

Although the Wood-Ljungdahl pathway, a wide-spread pathway for CO2 fixation in anaerobic microorganisms, was elucidated in the thermophilic acetogenic bacterium Moorella thermoacetica, still relatively little is known about the enzymes involved in hydrogen oxidation, CO2 fixation, energy conservation and the role of quinones and cytochromes. Here, we have used transcriptomics, enzyme assays and genome analyses to identify missing links. NADPH, generated by a [FeFe] hydrogenase, is the reductant for CO2 reduction to formate, a key reaction in CO2 fixation. This reaction is slightly endergonic under standard conditions but becomes thermodynamically feasible at high environmental H2 concentrations. In addition, formate is taken out of equilibrium by a formate dehydrogenase that potentially forms a complex with an energy-converting hydrogenase (Fdh-Ech), a novel respiratory enzyme in acetogens. Under low H2 concentrations, the complex can drive the reverse, endergonic reaction. In addition, we postulate a formate cycle involving a periplasmic, cytochrome b-containing formate dehydrogenase. A NADH dehydrogenase-like enzyme that uses reduced ferredoxin instead of NADH to reduce menaquinone is also involved in energy conservation. The data are summarised in a comprehensive metabolic and bioenergetic model of acetogenesis from H2 + CO2 and CO in M. thermoacetica.

A tungsten-specific maturation pathway governs cofactor assembly of a CO2-reducing formate dehydrogenase in Methylorubrum extorquens.

Chiral amines as pivotal intermediates in organic synthesis are widely utilized in the production of pharmaceuticals, fine chemicals, and bioactive molecules, holding significant industrial value. The asymmetric synthesis of chiral amines has always gained great attention. NAD(P)H-dependent imine reductases (IREDs) with wide substrate ranges, high activity, and high enantiomeric selectivity can be used for asymmetric reduction of imines to chiral amines. Our study aims to develop an efficient and stable immobilized dual-enzyme system through the fusion expression of imine reductase and formate dehydrogenase, in order to address the challenge of coenzyme regeneration and enhance catalytic efficiency, thereby providing a novel strategy for the green synthesis of chiral amines. The constructed system was applied to catalyze the asymmetric reduction of 1-methyl-3,4-dihydroisoquinoline to synthesize (S)-1-methyl-1,2,3,4-tetrahydroisoquinoline. Comparative analysis indicated that the catalytic efficiency of this fusion expression system exceeded that of both co-expression and standalone dual-enzyme systems. Furthermore, mesoporous silica nanoflowers were utilized as carriers to immobilize the fusion-expressed dual enzymes through a covalent method. In the case of covalent binding duration of 1.5 h and an initial enzyme concentration of 2.5 mg/mL, the protein loading achieved 193.2 mg/g. The immobilized enzymes demonstrated excellent pH, thermal, and storage stability. When the immobilized enzymes were employed to catalyze asymmetric reduction reactions of other cyclic imines, such as 1-ethyl-3,4-dihydroisoquinoline, 5-phenyl-3,4-dihydro-2H-pyrrole, 2,3,3-trimethyl-3H-indole, 2,3,3,5-tetramethylindole, and myosmine, the conversion rates exceeded 95%, and the values of e.e. surpassed 96%. The data confirm the application potential of the immobilized fusion enzymes in the green and efficient synthesis of chiral amines. Our study provides a novel strategy for the industrial biosynthesis of chiral amines, and the developed fused-enzyme immobilization approach holds significant theoretical and practical value for addressing common technical challenges in cofactor-dependent biocatalytic processes.

Integrating synthetic light-harvesting materials with biological CO2-fixing catalysts offers a promising route to efficient and selective solar-to-chemical conversion under mild conditions. However, progress remains limited by the lack of photocatalytic materials that combine biocompatibility, strong electronic coupling with biocatalysts, high biocatalyst loading capacity, and facile product separation. Here we introduce an organic semiconducting hydrogel synthesized from a rationally designed conjugated polyelectrolyte featuring visible-light absorption, water-processability, and covalent cross-linkability. The resulting macroporous, positively charged hydrogel scaffold immobilizes both microbes and enzymes, promoting intimate abiotic-biotic interactions throughout the three-dimensional hydrogel matrix. This platform supports two distinct modes of sacrificial CO2 reduction: mediated electron transfer via photogenerated H2 to drive acetate synthesis in the microbe Clostridium ljungdahlii, and direct electron transfer from photoexcited polymer domains to the isolated enzyme formate dehydrogenase for formate synthesis. By coupling the molecular programmability of organic semiconductors with the selectivity of biocatalysts, this work establishes a versatile class of soft biohybrid materials for solar fuel production through semiartificial photosynthesis.

The development of sustainable technologies for converting CO2 into value-added chemicals using solar energy remains a critical challenge. We presented a biohybrid system integrating Z-scheme photocatalysts with engineered microbial consortia for light-driven succinic acid production from CO2 and H2O without sacrificial agents. The system combined Escherichia coli biofilms expressing formate dehydrogenase for photoelectrochemical CO2 reduction to formate with adaptively evolved Vibrio natriegens for formate upgrading to succinic acid. This configuration achieved 0.06 mM succinic acid in 6 h with an apparent quantum yield of 0.154%. The biofilms maintained conformal contact with photocatalysts through four operational cycles, while V. natriegens viability increased by 11% owing to the formate provided by the biohybrid sheet. Further, isotopic tracing revealed approximately 20% carbon incorporation from CO2. This work establishes a sustainable platform for solar-driven multicarbon synthesis through rational integration of photocatalysis and synthetic microbial consortia.

The methylotrophic yeast Komagataella phaffii is a premier host for recombinant protein (rProt) production, which traditionally relies on methanol induction of the alcohol oxidase 1 promoter (PAOX1). However, the flammability and associated industrial limitations of methanol have motivated the search for methanol-free induction systems. We recently demonstrated that disruption of formate dehydrogenase gene (FDH) in K. phaffii allows endogenous formate derived from tetrahydrofolate (THF)-mediated C1 metabolism to induce PAOX1 without the addition of external inducers. Therefore, we hypothesized that increasing intracellular formate production by enhancing serine biosynthesis could further improve promoter induction and rProt productivity. Overexpression of SER3, encoding 3-phosphoglycerate dehydrogenase, the rate-limiting enzyme in serine synthesis, significantly increased PAOX1-driven expression of an intracellular reporter protein (eGFP) and secreted glucose oxidase (Gox) from Aspergillus niger without compromising cell fitness. Enhanced formate accumulation and stronger PAOX1 induction were observed in both microbioreactor and bioreactor cultivations using sorbitol or glycerol-sorbitol mixtures. In GOX-mSER3 strain grown in bioreactor, SER3 overexpression led to a 30% increase in specific Gox activity compared with that of the parental FdhKO strain. This study provides a cost-effective metabolic engineering strategy for methanol-free, self-inducible expression systems in K. phaffii based on PAOX1, enabling safer and more sustainable industrial rProt production.

Mo/W-dependent formate dehydrogenases (Fdhs) catalyze the reversible reduction of CO2 to formate and are key biocatalysts with high potential for CO2 capture/conversion technologies. Although previous studies have suggested the presence of two substrate-access tunnels in Fdhs, experimental evidence for CO2-specific pathways has been lacking. Here, we present an integrated study of Nitratidesulfovibrio vulgaris FdhAB combining crystallography, molecular dynamics simulations, mutagenesis, and kinetic assays. NvFdhAB crystals pressurized with Kr, O2, and CO2 were used to map gas diffusion routes and uncovered a substrate-retention site consistently occupied by small molecules in multiple crystal structures. Our results indicate that both substrates mostly use the main tunnel to reach this retention site, but H2O and CO2 can also enter through a novel side branch before following a shared route to the buried W active site. The retention site, located at the junction of both tunnels, plays a synergistic role in enhancing CO2 reduction by increasing substrate concentration near the catalytic center, thereby improving catalytic efficiency. Notably, variants affecting this site showed a selective effect for CO2 reduction, with no impact on formate oxidation. These findings provide experimental evidence of a CO2-specific pathway and identify structural determinants underpinning efficient CO2 reduction in this enzyme family.

Four formate dehydrogenases (FDHs) from Pseudomonas sp. 101, Myceliophthora thermophila, Chaetomium thermophilum, and Ogataea parapolymorpha were recombinantly produced, purified, and characterized to investigate their catalytic properties and reaction mechanisms. The enzymes were studied for their ability to oxidize formate to carbon dioxide (CO2) coupled with NAD+ reduction. In contrast, their CO2 reduction activity was undetectable under the tested conditions. Oxidative reactions revealed significant differences in catalytic efficiency and substrate specificity, prompting further investigation through molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) ONIOM calculations. Structural models were derived from high-resolution structural data available for enzymes from Pseudomonas sp. 101 (pseFDH) and Chaetomium thermophilum (ctFDH) and extended to all four variants. Comparative analyses of the transition states revealed distinct interaction patterns within the active sites, allowing us to discriminate between high- and low-performing catalysts, in full agreement with the experimental kcat values. These findings provide a mechanistic rationale for the observed disparities in catalytic performance and offer structural insights into the determinants of FDH activity. Notably, ctFDH emerged as a potential candidate for the development of CO2-reducing reactions, with QM/MM data guiding the rational design of transition-state stabilizing mutations.

Bioelectrocatalytic CO2 reduction offers a sustainable route for CO2 bioconversion, yet remains limited by interfacial-intramolecular electron transfer and oxygen sensitivity. Here, we mine a formate dehydrogenase from Shewanella oneidensis MR-1 (SoFdhAB) featuring completely oxygen tolerant and direct-electron-transfer (DET) electrocatalytic performances. Cryo-electron microscopy (Cryo-EM) analysis reveals an intramolecular electron highway comprising five [4Fe-4S] clusters, a regional face-face contact facilitating interfacial ET, and a unique oxygen resistance mechanism different from inactivation-activation. By acquiring a beneficial variant SoFdhAB-Y94S, a direct bioelectrocatalytic CO2 reduction system is constructed, accumulating 2.88 ± 0.03 mmol formate in 64 hours with a steady rate of 45.3 ± 0.5 μmol h-1 cm-2 and a Faradaic efficiency of 93.1 ± 5.2%. The merits of oxygen tolerance and efficient (electro)catalytic property endow SoFdhAB a robust enzyme adopted in potential application scenarios, and the inherent DET capability may inspire the interfacial engineering of other oxidoreductases.

Staphylococcus aureus is an opportunistic pathogen that can form biofilms and is known for its persistence in causing infections. This study aims to characterise the transcriptional mechanisms underlying biofilm formation in both methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains. Total RNA was extracted from cells grown under biofilm and planktonic conditions and subjected to RNA sequencing on the Illumina HiSeq 2500. Differential expression analysis was performed using DESeq2 and functional enrichment was assessed through Kyoto Encyclopedia of Genes and Genomes enrichment pathway analyses. Transcriptome analysis identified 2,809 genes in MRSA (612 DEGs: 552 upregulated, 60 downregulated) and 2,744 genes in MSSA (66 DEGs: 29 upregulated, 37 downregulated) expressed genes in MRSA and MSSA biofilm cells, respectively. The genes that are significantly differentially expressed in biofilms (log2 fold change > 1, p-value < 0.05) include 221 genes in MRSA and 12 in MSSA. MRSA biofilms showed a significant upregulation of known genes associated with biofilm formation, including polysaccharide adhesin synthesis (icaADBC), fibronectin-binding (fnbA), extracellular matrix-binding protein (Embp) and surface protein C (sasC). In contrast, the upregulated genes in MSSA biofilms highlight aspects of metabolism (ferredoxin, formate dehydrogenase), transport (aquaporin, ABC transporter) and virulence staphylococcal secretory antigen A (ssaA). MRSA and MSSA exhibit distinct gene expression profiles in their biofilm cells, suggesting that biofilm formation in MRSA and MSSA is strain-specific.

Formaldehyde detoxification by the moss Racomitrium japonicum.

The production of biofuels by bacterial fermentation receives sustained attention due to the need to develop novel circular and sustainable technologies. Clostridium beijerinckii produces both hydrogen (H2) and carbon-based biofuels acetone, butanol and ethanol (ABE solvents). H2 metabolism in C. beijerinckii is complex and mostly unexplored. Seven hydrogenase genes are contained in the genome, but their exact physiological role is unknown. Here, we report on the characterisation of a novel heterotetrameric soluble enzyme complex composed of an [FeFe]-hydrogenase component stably bound to a formate dehydrogenase subunit, which we name CbFdh/Hyd. We show that the four subunits form a stable complex that can be conveniently overexpressed and purified recombinantly. CbFdh/Hyd is highly sensitive to atmospheric oxygen and displays reversible catalytic features, including H2 evolution, H2 uptake, formate oxidation and the ability to split formate into H2 and CO2 (formate hydrogen lyase activity, FHL) as well as the opposite reaction, H2-driven CO2 reduction (HDCR). CbFdh/Hyd displays functional and spectroscopic features very similar to Fdh/Hyd complexes previously described in acetogens, suggesting that this enzyme is at the basis of the previously reported unconventional ability of C. beijerinckii to fix CO2 into acetate and butyrate. CbFdh/Hyd could also represent a key player in H2 production metabolism by degrading formate produced from the decarboxylation of pyruvate.

The mechanism of selective specificity of oxidoreductases to NAD+ or NADP+ and the ability to change the coenzyme specificity of these enzymes are some of the most important fundamental and applied problems. The first work on the switch in the coenzyme specificity from NADP+ to NAD+ was performed in 1990 for glutathione reductase. In1993, formate dehydrogenase (FDH, EC1.2.1.2) from the methylotrophic bacterium Pseudomonassp.101 (PseFDH) became the first oxidoreductase whose coenzyme specificity was changed in the opposite direction- from NAD+ to NADP+. Mutant NADP+-specific FDHs are extensively used in fine organic synthesis (including production of chiral compounds). The switch in the coenzyme specificity from NAD+ to NADP+ in FDHs is achieved by substituting amino acids at positions 198, 221, 222, 260, 379, and 380 (numbering according to PseFDH); however available data do not allow the interpretation of the exact role of each individual substitution. Since2010, five natural NADP+-dependent FDHs have been found. In2015-2024, three 3Dstructures for two natural and four 3Dstructures for two mutant NADP+-specific FDHs have appeared in the Protein Data Bank (PDB). In this review, we briefly discussed the general principles of coenzyme specificity based on the experimental and modeled FDH structures and performed a detailed analysis of the type and arrangement of residues at positions corresponding to His379 and Ser380 in PseFDH, whose role in NADP+ binding is still debated.

High specificity of MsmCAR toward 4-hydroxyvaleric acid enables efficient 1,4-pentanediol production from biomass-derived levulinic acid.