Mechanism of Action of Formate Dehydrogenases.

The molybdenum- and tungsten-containing formate dehydrogenases from a variety of microorganisms catalyze the reversible interconversion of formate and CO2; several, in fact, function as CO2 reductases in the reverse direction under physiological conditions. CO2 reduction catalyzed by these enzymes occurs under mild temperature and pressure rather than the elevated conditions required for current industrial processes. Given the contemporary importance of remediation of atmospheric CO2 to address global warming, there has been considerable interest in the application of these enzymes in bioreactors. Equally important, understanding the detailed means by which these biological catalysts convert CO2 to formate, a useful and easily transported feedstock chemical, might also inspire the development of a new generation of highly efficient, biomimetic synthetic catalysts. Here we have examined the ability of the FdsDABG formate dehydrogenase from Cupriavidus necator to catalyze the exchange of solvent oxygen into product CO2 during the course of formate oxidation under single-turnover conditions. Negligible incorporation of 18O is observed when the experiment is performed in H218O, indicating that bicarbonate cannot be the immediate product of the enzyme-catalyzed reaction, as previously concluded. These results, in conjunction with the observation that the reductive half-reaction exhibits mildly acid-catalyzed rather than base-catalyzed chemistry, are consistent with a reaction mechanism involving direct hydride transfer from formate to the enzyme's molybdenum center, directly yielding CO2. Our results are inconsistent with any mechanism in which the initial product formed on oxidation of formate is bicarbonate.

Rational Design of Formate Dehydrogenase for Enhanced Thermal Stability and Catalytic Activity in Bioelectrocatalysis.

Formate dehydrogenase can be utilized as a biocatalyst in the bioelectrocatalysis of converting CO2 into formic acid. However, its industrial application has been hindered by limited thermal stability. This study successfully obtained a mutant (D533S/E684I) with enhanced thermal stability and catalytic activity through the rational design of flexible regions. The mutant exhibited a half-life (t1/2) 1.5 times longer than the wild type (WT) at 35 °C, along with a specific enzyme activity 7.46 times higher than that of the WT. Additionally, the catalytic efficiency (kcat/Km value) of the mutant toward the substrate was 2.72 s-1·mM-1, representing a 19.4-fold increase compared to the WT (0.14 s-1·mM-1). Formic acid production reached 53.4 mM through bioelectrocatalysis after 10 h, utilizing the mutant as the biocatalyst. Molecular dynamics simulations and structural analysis were employed to investigate the molecular mechanisms behind the enhanced thermal stability and activity. The displacement of a highly flexible region in the mutant may counteract the stability-activity trade-off. This study proposed a method for improving both thermal stability and activity in enzyme evolution.

Immobilization of cross-linked enzymes aggregates on hierarchical covalent organic frameworks: Highly stable chemoenzymatic nanoreactor for asymmetric synthesis of optically active halohydrins.

Organometallic catalyst is extensively applied for the non-enzymatic regeneration of nicotinamide adenine dinucleotide (phosphate) cofactors, but suffering from the mutual inactivation with the enzymes in one pot. The spatially separated immobilization of organometallic catalyst and enzymes on suitable carriers not only can reduce their mutual inhabitation but also can enhance their reusability. Here in this work, we present a hierarchical porous COFs (HP-TpBpy) that incorporated with [(Cp*RhCl2]2 to generate the metalized COF, Rh-HP-TpBpy. The obtained Rh-HP-TpBpy exhibited superior performance in nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration using formate as the hydride donor, significantly outperforming the natural formate dehydrogenases in cofactor preference toward NADP+. Subsequently, the Lactobacillus fermentum short-chain dehydrogenase/reductase 1 (LfSDR1) was then cross-linked into enzyme aggregates (CLEA) and immobilized on hierarchical Rh-HP-TpBpy, achieving the integrated chemoenzymatic catalyst, LfSDR1@Rh-HP-TpBpy, which can catalyze the chemoenzymatic reduction of halogenated aryl ketones and give the corresponding optically active halohydrins with high conversion and enantiomeric excess (ee) value up to 99 %. The LfSDR1@Rh-HP-TpBpy also exhibits largely enhanced stability compared with the free LfSDR1 and the CLEAs-LfSDR1, enabling its excellent reusability.

Surface Au-H Species as Self-Generated Prosthetic Groups of a Formate Dehydrogenase-like Au Nanozyme to Engineer Multienzymatic Activities.

Although the past decade has witnessed a rapid development of oxidoreductase-mimicking nanozymes, the mimicry of cofactors that play key roles in mediating electron and proton transfer remains limited. This study explores how surface Au-H species conjugated to Au nanoparticles (NPs) that imitate formate dehydrogenase (FDH) can serve as cofactors, analogous to NADH in natural enzymes, offering diverse possibilities for FDH-mimicking Au nanozymes to mimic various enzymes. Once O2 is present, Au-H species assist Au NPs to complete the on-demand H2O2 generation for cascade reactions. Alternatively, when oxidizing organic molecules are introduced as substrates, Au-H species confer nitro reductase- and aldehyde reductase-like activities on Au NPs under anaerobic conditions. Furthermore, similar to the dehydrogenase-NADH complex, Au NPs possessing Au-H species are gifted with esterase-like activity for ester hydrolysis. By revealing that Au-H species are prosthetic groups for FDH-mimicking Au nanozymes, this work may inspire explorations into future self-generated cofactor mimics for nanozymes, thereby circumventing the need for exogenous cofactors.

One-pot enantioselective synthesis of chiral phenyllactic acids by combining stereocomplementary d- and l-lactate dehydrogenases with multi-enzyme expression fine-tuning.

Chiral phenyllactic acid (PLA) is a new type of antiseptic agent and a valuable precursor for active ingredients in pharmaceuticals and agrochemicals. In this study, we designed a multi-enzyme cascade that combined stereocomplementary d- and l-lactate dehydrogenases with threonine aldolase, phenylserine dehydratase, and formate dehydrogenase for the one-pot conversion of achiral glycine and benzaldehyde to synthesize d-PLA and l-PLA. To overcome the imbalance of multi-enzymes in a single cell, two enzyme modules, overexpressing four enzymes, were assembled in Escherichia coli cells to construct whole-cell catalysis systems (WCCSs). Furthermore, by optimizing reaction conditions and components, recombinant E. coli (WCCS 26) was able to produce 100 mM d-PLA with >99 % ee using a fed-batch strategy, while E. coli (WCCS 60) produced 47.2 mM l-PLA with >99 % ee. This study presents a sustainable and efficient method for synthesizing chiral PLAs from food-grade achiral starting materials.

Mechanoresponsive Protein Crystals for NADH Recycling in Multicycle Enzyme Reactions.

NAD(H)-dependent enzymes play a crucial role in the biosynthesis of pharmaceuticals and fine chemicals, but the limited recyclability of the NAD(H) cofactor hinders its more general application. Here, we report the generation of mechano-responsive PEI-modified Cry3Aa protein crystals and their use for NADH recycling over multiple reaction cycles. For demonstration of its practical utility, a complementary Cry3Aa protein particle containing genetically encoded and co-immobilized formate dehydrogenase for NADH regeneration and leucine dehydrogenase for catalyzing the NADH-dependent l-tert-leucine (l-tert-Leu) biosynthesis has been produced. When combined with the PEI-modified Cry3Aa crystal, the resultant reaction system could be used for the efficient biosynthesis of l-tert-Leu for up to 21 days with a 10.5-fold improvement in the NADH turnover number.

Electrocatalysis of CO2 Reduction by Immobilized Formate Dehydrogenase without a Metal Redox Center.

Nicotinamide adenine dinucleotide-dependent formate dehydrogenase from Candida boidinii was immobilized in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/cholesterol floating lipid bilayer on the gold surface as a biocatalyst for electrochemical CO2 reduction. We report that, in contrast to common belief, the enzyme can catalyze the electrochemical reduction of CO2 to formate without the cofactor protonated nicotinamide adenine dinucleotide. The electrochemical data indicate that the enzyme-catalyzed reduction of CO2 is diffusion-controlled and is a reversible reaction. The orientation and conformation of the enzyme were investigated by surface-enhanced infrared reflection absorption spectroscopy. The α-helix of the enzyme adopts an orientation nearly parallel to the surface, bringing its active center close to the gold surface. This orientation allows direct electron transfer between CO2 and the gold electrode. The results in this paper provide a new method for the development of enzymatic electrocatalysts for CO2 reduction.

Construction of Multi-Enzyme Integrated Catalysts for Deracemization of Cyclic Chiral Amines.

Multi-enzyme cascade catalysis has become an important technique for chemical reactions used in manufacturing and scientific study. In this research, we designed a four-enzyme integrated catalyst and used it to catalyse the deracemization reaction of cyclic chiral amines, where monoamine oxidase (MAO) catalyses the enantioselective oxidation of 1-methyl-1,2,3,4-tetrahydroisoquinoline (MTQ), imine reductase (IRED) catalyses the stereo selective reduction of 1-methyl-3,4-dihydroisoquinoline (MDQ), formate dehydrogenase (FDH) is used for the cyclic regeneration of cofactors, and catalase (CAT) is used for decomposition of oxidative reactions. The four enzymes were immobilized via polydopamine (PDA)-encapsulated dendritic organosilica nanoparticles (DONs) as carriers, resulting in the amphiphilic core-shell catalysts. The hydrophilic PDA shell ensures the dispersion of the catalyst in water, and the hydrophobic DON core creates a microenvironment with the spatial confinement effect of the organic substrate and the preconcentration effect to enhance the stability of the enzymes and the catalytic efficiency. The core-shell structure improves the stability and reusability of the catalyst and rationally arranges the position of different enzymes according to the reaction sequence to improve the cascade catalytic performance and cofactor recovery efficiency.

Structural and biochemical characterization of the M405S variant of Desulfovibrio vulgaris formate dehydrogenase.

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO2 to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine Sδ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO2 reduction, together with an increased sensitivity to oxygen inactivation.

Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

Structure of recombinant formate dehydrogenase from Methylobacterium extorquens (MeFDH1).

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Redox potentials elucidate the electron transfer pathway of NAD+-dependent formate dehydrogenases.

Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (FeS) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined FeS clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis.

Study of the structure-function relationship of formate dehydrogenase- an important enzyme for Staphylococcus aureus biofilms by rational design.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Staphylococcus aureus (SauFDH) plays an important role in the vital activity of this bacterium, especially in the form of biofilms. Understanding its mechanism and structure-function relationship can help to find special inhibitors of this enzyme, which can be used as medicines against staphylococci. The gene encoding SauFDH was successfully cloned and expressed in our laboratory. This enzyme has the highest kcat value among the described FDHs and also has a high temperature stability compared to other enzymes of this group. That is why it can also be considered as a promising catalyst for NAD(P)H regeneration in the processes of chiral synthesis with oxidoreductases. In this work, the principle of rational design was used to improve SauFDH catalytic efficiency. After bioinformatics analysis of the amino acid sequence in combination with visualization of the enzyme structure (PDB 6TTB), 9 probable catalytically significant positions 119, 194, 196, 217-219, 246, 303 and 323 were identified, and 16 new mutant forms of SauFDH were obtained and characterized by kinetic experiments. The introduction of the mentioned substitutions in most cases leads to a decrease in stability at high temperatures and an increase at low temperatures. Substitutions in positions 119 and 194 lead to a decreasing of KMNAD+. A consistent decrease in the Michaelis constant in the Ile-Val-Ala-Gly series at position 119 of SauFDH is shown. KMNAD+ of mutant SauFDH V119G decreased by 27 times compared to the wild-type enzyme. After substitution Phe194Val KMNAD + decreased by 3.5 times. The catalytic constant for this mutant form practically did not change. For this mutant form, an increase in catalytic efficiency was demonstrated through the use of a multicomponent buffer system.

A Multienzyme Cascade Pathway Immobilized in a Hydrogen-Bonded Organic Framework for the Conversion of CO2.

The reduction of carbon dioxide to valuable chemicals through enzymatic processes is regarded as a promising approach for the reduction of carbon dioxide emissions. In this study, an in vitro multi-enzyme cascade pathway is constructed for the conversion of CO2 into dihydroxyacetone (DHA). This pathway, known as FFFP, comprises formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH), formolase (FLS), and phosphite dehydrogenase (PTDH), with PTDH serving as the critical catalyst for regenerating the coenzyme NADH. Subsequently, the immobilization of the FFFP pathway within the hydrogen-bonded organic framework (HOF-101) is accomplished in situ. A 1.8-fold increase in DHA yield is observed in FFFP@HOF-101 compared to the free FFFP pathway. This enhancement can be explained by the fact that within FFFP@HOF-101, enzymes are positioned sufficiently close to one another, leading to the elevation of the local concentration of intermediates and an improvement in mass transfer efficiency. Moreover, FFFP@HOF-101 displays a high degree of stability. In addition to the establishment of an effective DHA production method, innovative concepts for the tailored synthesis of fine compounds from CO2 through the utilization of various multi-enzyme cascade developments are generated by this work.

Leveraging liquid-liquid phase separation and volume modulation to regulate the enzymatic activity of formate dehydrogenase.

Engineering of reaction media is an exciting alternative for modulating kinetic properties of biocatalytic reactions. We addressed the combined effect of an aqueous two-phase system (ATPS) and high hydrostatic pressure on the kinetics of the Candida boidinii formate dehydrogenase-catalyzed oxidation of formate to CO2. Pressurization was found to lead to an increase of the binding affinity (decrease of KM, respectively) and a decrease of the turnover number, kcat. The experimental approach was supported using thermodynamic modeling with the electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. The ePC-SAFT was able to quantitatively predict the KM-values of the substrate in both phases at 1 bar as well as up to a pressure of 1000 bar. The framework presented enables significant advances in bioprocess engineering, including the design of processes with significantly fewer experiments and trial-and-error approaches.

Redox Characterization of the Complex Molybdenum Enzyme Formate Dehydrogenase from Cupriavidus necator.

The oxygen-tolerant and molybdenum-dependent formate dehydrogenase FdsDABG from Cupriavidus necator is capable of catalyzing both formate oxidation to CO2 and the reverse reaction (CO2 reduction to formate) at neutral pH, which are both reactions of great importance to energy production and carbon capture. FdsDABG is replete with redox cofactors comprising seven Fe/S clusters, flavin mononucleotide, and a molybdenum ion coordinated by two pyranopterin dithiolene ligands. The redox potentials of these centers are described herein and assigned to specific cofactors using combinations of potential-dependent continuous wave and pulse EPR spectroscopy and UV/visible spectroelectrochemistry on both the FdsDABG holoenzyme and the FdsBG subcomplex. These data represent the first redox characterization of a complex metal dependent formate dehydrogenase and provide an understanding of the highly efficient catalytic formate oxidation and CO2 reduction activity that are associated with the enzyme.

Engineering of formate dehydrogenase for improving conversion potential of carbon dioxide to formate.

Formate dehydrogenase (FDH) is a D-2-hydroxy acid dehydrogenase, which can reversibly reduce CO2 to formate and thus act as non-photosynthetic CO2 reductase. In order to increase catalytic efficiency of formate dehydrogenase for CO2 reduction, two mutants V328I/F285W and V354G/F285W were obtained of which reduction activity was about two times more than the parent CbFDHM2, and the formate production from CO2 catalyzed by mutants were 2.9 and 2.7-fold higher than that of the parent CbFDHM2. The mutants had greater potential in CO2 reduction. The optimal temperature for V328I/F285W and V354G/F285W was 55 °C, and they showed increasement of relative activity under 45 °C to 55 °C compared with parent. The optimal pH for the mutants was 9.0, and they showed excellent stability in pH 4.0-11.5. The kcat/Km values of mutants were 1.75 times higher than that of the parent. Then the molecular basis for its improvement of biochemical characteristics were preliminarily elucidated by computer-aided methods. All of these results further established a solid foundation for molecular modification of formate dehydrogenase and CO2 reduction.

Structural analysis of wild-type and Val120Thr mutant Candida boidinii formate dehydrogenase by X-ray crystallography.

Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential application in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of wild-type CbFDH at cryogenic and ambient temperatures, as well as that of the Val120Thr mutant at cryogenic temperature, determined at the Turkish Light Source `Turkish DeLight'. The structures reveal new hydrogen bonds between Thr120 and water molecules in the active site of the mutant CbFDH, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, these findings provide invaluable insights into future protein-engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.

Creating NADP+ -Specific Formate Dehydrogenases from Komagataella phaffii by Enzymatic Engineering.

Most natural formate dehydrogenases (FDHs) exhibit NAD+ specificity, making it imperative to explore the engineering of FDH cofactor specificity for NADPH regeneration systems. The endogenous FDH of Komagataella phaffii (K. phaffii), termed KphFDH, is a typical NAD+ -specific FDH. However, investigations into engineering the cofactor specificity of KphFDH have yet to be conducted. To develop an NADP+ -specific variant of KphFDH, we selected D195, Y196, and Q197 as mutation sites and generated twenty site-directed variants. Through kinetic characterization, KphFDH/V19 (D195Q/Y196R/Q197H) was identified as the variant with the highest specificity towards NADP+ , with a ratio of catalytic efficiency (kcat /KM )NADP+ /(kcat /KM )NAD+ of 129.226. Studies of enzymatic properties revealed that the optimal temperature and pH for the reduction reaction of NADP+ catalyzed by KphFDH/V19 were 45 °C and 7.5, respectively. The molecular dynamics (MD) simulation was performed to elucidate the mechanism of high catalytic activity of KphFDH/V19 towards NADP+ . Finally, KphFDH/V19 was applied to an in vitro NADPH regeneration system with Meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH/H227V). This study successfully created a KphFDH variant with high NADP+ specificity and demonstrated its practical applicability in an in vitro NADPH regeneration system.

The Reversible Electrochemical Interconversion of Formate and CO2 by Formate Dehydrogenase from Cupriavidus necator.

The bacterial molybdenum (Mo)-containing formate dehydrogenase (FdsDABG) from Cupriavidus necator is a soluble NAD+-dependent enzyme belonging to the DMSO reductase family. The holoenzyme is complex and possesses nine redox-active cofactors including a bis(molybdopterin guanine dinucleotide) (bis-MGD) active site, seven iron-sulfur clusters, and 1 equiv of flavin mononucleotide (FMN). FdsDABG catalyzes the two-electron oxidation of HCOO- (formate) to CO2 and reversibly reduces CO2 to HCOO- under physiological conditions close to its thermodynamic redox potential. Here we develop an electrocatalytically active formate oxidation/CO2 reduction system by immobilizing FdsDABG on a glassy carbon electrode in the presence of coadsorbents such as chitosan and glutaraldehyde. The reversible enzymatic interconversion between HCOO- and CO2 by FdsDABG has been realized with cyclic voltammetry using a range of artificial electron transfer mediators, with methylene blue (MB) and phenazine methosulfate (PMS) being particularly effective as electron acceptors for FdsDABG in formate oxidation. Methyl viologen (MV) acts as both an electron acceptor (MV2+) in formate oxidation and an electron donor (MV+•) for CO2 reduction. The catalytic voltammetry was reproduced by electrochemical simulation across a range of sweep rates and concentrations of formate and mediators to provide new insights into the kinetics of the FdsDABG catalytic mechanism.