The review analyzes recent advances, challenges, and practical applications in the field of enzymes within the framework of chemical enzymology and enzyme engineering. The achievements in the fundamental understanding of molecular mechanisms of the catalytic cycle of enzymatic reactions made using quantum mechanics/molecular mechanics methods with supercomputer technologies and bioinformatic approaches are considered. The design of protein biocatalysts with new properties is a fundamentally significant methodology of the bioengineering approach to solving practical problems, which is demonstrated by a number of examples. The increasing role of biocatalysis in medicine and biomedical research is illustrated by addressing the problems of antibiotic synthesis and overcoming antibiotic resistance of bacteria, mechanisms of neurodegenerative diseases and development of drugs to treat Alzheimer's disease, biocatalytic processes of DNA repair and the role of mechanisms of functioning of heme peroxidases in the human body. The use of enzymes to degrade endogenous and exogenous toxicants has been greatly developed in recent decades. The advances and problems of using enzymes in therapy and drug delivery are analyzed. The fundamental role of enzymes in modern analysis and diagnosis is noted. The review considers a new trend in the development of bioanalytical methods using aptamers, multi-analysis systems on biochips, surface-enhanced Raman scattering systems, and bioelectroanalysis. The bibliography includes 460 references.

NAD -dependent formate dehydrogenase from the bacterium Staphylococcus aureus (SauFDH) plays an important role in the vital activity of these bacteria. The gene encoding SauFDH was successfully cloned and expressed in our laboratory. Since this enzyme has the highest k value among the described FDHs and also has a high temperature stability compared to other proteins, it can be considered as a promising catalyst for regeneration of NAD(P)H. The main disadvantage of this enzyme are high K values. In this work, the principle of rational design was used to reduce K . As a result, 9 catalytically significant positions 119, 194, 196, 217-219, 246, 303, and 323 were identified, and 16 new mutant forms of SauFDH were obtained and fully characterized. Substitutions in positions 119 and 194 lead to an increase of K . In the Ile-Val-Ala-Gly line, position 119 tends to improve NAD binding. K of SauFDH V119G decreased by 27 times compared to the wild-type enzyme. K Phe194Val decreased by 3.5 times. The catalytic constant for this mutant form practically did not change. The use of a complex buffer increased the catalytic efficiency 6 times for the mutant with the Phe194Val substitution compared to wt-SauFDH in a single-component buffer.

NAD(P)+-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) catalyzes the oxidation of formate ion with the coupled reduction of NAD(P)+ to NAD(P)H. Previously, in our laboratory, a genetic construct was obtained with the soyfdh2 gene encoding isoenzyme 2 of formate dehydrogenase from soybean Glycine max (SoyFDH). In this construct the nucleotide sequence encoding the signal peptide responsible for the transport of the pro-enzyme into the mitochondria of plant cells (the SoyFDH_L enzyme) was deleted. In this work, a second variant of SoyFDH_S was obtained, in which, compared to SoyFDH_L, the sequence at the N-terminus was reduced and changed to mimic the N-terminus sequence in FDH from Pseudomonas sp.101 bacterium. Next, a sequence of six histidine residues (His-tag) was added to the N-terminus of the long and short forms of SoyFDH. All four SoyFDH variants were expressed in E. coli BL21(DE3)CodonPlus cells. These enzymes were purified, their kinetic parameters were determined, and thermal stability was studied. In the case of SoyFDH_L, which is similar to the natural form of the enzyme, both variants, with and without His-tag, the expression level is two times higher compared to the truncated variant. The addition of His-tag to the N-terminus of enzymes reduces the level of expression. Changing the sequence of the N-terminus, as well as introducing the His-tag sequence to the N-terminus, does not significantly affect thermal stability of the enzymes at temperatures of 50–56°C. However, due to the higher values of the activation enthalpy ΔH≠ of the thermal inactivation process, the shortened form at normal temperatures is 3 times more stable than the natural one. A comparison of the kinetic parameters of the two SoyFDH variants shows that the catalytic constants are the same, but the long version SoyFDH_L has lower values K_M^HCOO - , and the short version SoyFDH_S has lower K_M^NAD + values. The introduction of His-tag into the N-terminus of enzymes does not affect their kinetic parameters.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from methylotrophic bacterium Pseudomonas sp.101 (PseFDH) has one of the highest thermal stability among all known enzymes of this group. The introduction of a number of amino acid substitutions into PseFDH made it possible to obtain a multipoint mutant PseFDH SM4S enzyme with even higher temperature and chemical stability. Previously, we showed that the introduction of additional single point replacements S131A, or S160A, or E170D into PseFDH SM4S led to further stabilization of the enzyme. In this work, based on the PseFDH SM4S S131A mutant, new mutant FDHs obtained, in which, compared to PseFDH SM4S, we added double S131A/E170D (M2), triple S131A/S160A/E170D (M3) and quadruple S131A/S160A/E170D/S145A (PseFDH SM4A M3) amino acids replacements. The new PseFDH mutants were overexpressed in E. coli cells, purified and characterized. The S131A/E170D and S131A/S160A/E170D changes provided further improving thermal stability. The introduction of the S145A substitution into PseFDH SM4A M3 leads to a significant decrease in K_M^NAD^ + and K_M^HCOO^ - while maintaining the catalytic constant at the same level. This mutant form can be successfully used in NADH regeneration systems, as well as for the detection of NAD+ and formate in biological systems.

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from pathogenic bacterium Staphylococcus aureus (SauFDH) differs significantly from other FDHs both in terms of primary structure and catalytic properties. A distinctive feature of SauFDH is the highest (about 2.5–3 times) specific activity compared to other formate dehydrogenases. At the same time, SauFDH has high Michaelis constants for both substrates. Based on the analysis of three-dimensional structures and the alignment of amino acid sequences, replacements promising in terms of changing catalytic parameters were selected. The replacement of I220H resulted in an increase in K_M^NAD^ + ; the value of kcat has not changed. When T250H is replaced, an increase in K_M^NAD^ + is observed, kcat decreases from 20 to 13 s–1. The replacement of K368H led to a slight increase in K_M^NAD^ + , kcat decreased from 20 to 6 s–1. The introduction of TGA and AGA additional inserts in α-helix at the C-terminus of the enzyme led to an increase in K_M^NAD^ + and K_M^HCOO^ - . A bigger effect was observed for K_M^NAD^ + —the difference was more than 10 times. For mutant SauFDH with insertions kcat significantly reduced to 4 s–1. Similar results were observed for mutants with multipoint replacements. Thus, the C-terminal sequence has been shown to play an important role in the catalysis of SauFDH.

D-amino acid oxidase (DAAO) plays an important role in the functioning of both prokaryotes and eukaryotes. DAAO is increasingly being used in practice, including for the determination of D-amino acids in complex samples, involving human tissues and fluids. There are generally two types of DAAO in all organisms. The first type is an enzyme highly specific for D-aspartate and has its own name D-aspartate oxidase (DASPO). DAAO of the second type is characterized by a wide spectrum of substrate specificity, with preference for one or another D-amino acid varying from source to source. The activity of DAAO with a large number of substrates greatly complicates the selective determination of a particular D-amino acid. The problem is often solved by choosing an enzyme that, under the conditions of analysis, has low or no activity with other D-amino acids present in the sample. For the convenience of selecting a particular enzyme, we have collected and analyzed literature data on the catalytic parameters of known DAAOs with the most important D-amino acids. In addition, similar data are presented for novel recombinant DAAOs from the methylotrophic yeast Ogataea parapolymorpha DL-1. Analysis of the data shows that, with the D-amino acid series, the new OpaDASPO and OpaDAAO have the highest catalytic parameters.

Our earlier annotation of the genome of the yeast Ogataea parapolymorpha DL-1 made it possible to identify five genes of potential D-amino acids oxidases. All opadaao1–opadaao5 genes were cloned and expressed in E. coli. Four OpaDAAO1-OpaDAAO4 enzymes were obtained in highly purified form and their catalytic properties were studied. It was found that among all DAAO described in the literature, the enzyme OpaDAAOl has the highest catalytic constant kcat with D-Ala, which makes it promising for practical applications. However, in addition to good catalytic parameters, effective application of the enzyme in practice requires stability and knowledge of the inactivation mechanism, including at elevated temperatures. In this paper, we study the effect of elevated temperatures on the stability of OpaDAAOl. The enzyme is shown to have higher thermal stability than the majority of other D-amino acid oxidases. The kinetics of OpaDAAOl inactivation at different temperatures, at the initial concentrations of the enzyme, and in the presence of exogenous FAD are studied. A possible kinetic scheme of inactivation is proposed based on the data obtained.

NAD(P)+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) catalyzes the simplest reaction from chemical and biological points of view, oxidation of formate-ion to carbon dioxide coupled to NAD(P)+ reduction to yield NAD(P)H. Advances in the life sciences have shown that this reaction plays an extremely important role in a wide variety of organisms. The areas and types of practical applications of FDH are also permanently expanding. The review analyzes the key steps in the development of our knowledge on the role of formate dehydrogenase in living systems. Achievements in creation of highly efficient catalysts based on FDH for classic biotechnology as well as for new areas are also considered. The importance of a correct selection of the starting FDH form for the purpose of a biocatalyst design with required properties with minimal costs is demonstrated. The prospects for the use of FDH for CO2 fixation of CO2 are discussed.

Phenylacetone monooxygenase from Thermobifida fusca (EC 1.14.13.92, PAMO) belongs to the Baeyer–Villiger family of monooxygenases and catalyzes the oxidation of various aromatic ketones to the corresponding esters using NADPH as a cofactor. In this study we analyzed the structure of the cofactor binding site, selected the most important amino acid residues for recognition of the phosphate group of the cofactor, and simulated enzyme structures with amino acid substitutions that could potentially lead to a change in the coenzyme specificity of the enzyme from NADPH to NADH. Based on the modeling, the most promising amino acid substitutions, T218D, T218E, K336A, and K336R, were proposed. Using site-directed mutagenesis we obtained genetic constructs containing genes encoding PAMOs with the corresponding amino acid substitutions, and the enzymes were expressed and purified. The resulting mutant PAMOs are able to bind NADH, but lack the ability to catalyze the oxidation of benzylacetone in the presence of NADH and show a deterioration of the Michaelis constants for NADPH. The catalytic constants of the mutant enzymes studied decrease slightly, and remain within the allowed experimental error.

Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC 1.2.1.2) is a research model for the elucidation of the catalytic mechanism of 2-oxyacid D-specific dehydrogenases enzyme superfamily. The enzyme is actively used for regeneration of the reduced form of NAD(P)H in chiral synthesis with oxidoreductases. A multi-point mutant PseFDH SM4S with an improved thermal and chemical stability has been prepared earlier in this laboratory. To further improve the properties of the mutant, additional single-point replacements have been introduced to generate five new PseFDH mutants. All new enzymes have been highly purified, and their kinetic properties and thermal stability studied using analysis of thermal inactivation kinetics and differential scanning calorimetry. The E170D amino acid change in PseFDH SM4S shows an increase in thermal stability 1.76- and 10-fold compared to the starting mutant and the wild-type enzyme, respectively.

Once you have missed the first button …, you'll never manage to button up Johann Wolfgang von Goethe Formate oxidation is a final step of methanol oxidation in methylotrophic prokaryotes and is important for detoxification of formate in other organisms. The structural mechanism of the formate dehydrogenase (FDH) of Pseudomonas sp. 101 has been studied for about 30 years. In the active center of FDH, the oxidation of formic acid into carbon dioxide in a NAD+-dependent way takes place. Residues that form the active center of that enzyme, as well as those that form the so-called substrate channel, are engaged in the catalytic cycle. Our study allowed to characterize a new residue, Tyr102, involved in the work of the enzyme. This residue is located in the outer neck of the substrate channel (at the beginning of the path of the substrate to the active center) and acts as a "button" which connects two enzyme domains into an active, "buttoned up" conformation. Our study of the kinetic parameters of mutant enzymes has shown that Tyr102Phe substitution leads to an approximately 80-fold increase of the Michaelis constant relative to the native enzyme, unlike Phe311Trp and Phe311Tyr substitution of neighboring residue Phe311. Our analysis of the Tyr102Phe mutant in the open conformation by X-ray crystallography has shown that its overall fold remains almost the same as that of the native enzyme. Molecular dynamics simulations of the ternary complexes of the native FDH enzyme and its Tyr102Phe mutant showed that Tyr102Phe substitution results in the loss of an interdomain hydrogen bond between the Tyr102 and Gln313 residues, which, in turn, destabilizes the closed conformation and affects the isolation of the FDH active site from water molecules. Our structural investigations have shown that Tyr102Phe replacement also leads to the destruction of interdomain contacts of Phe102 with Phe311, Pro312 residues, and decreases the stability of the Leu103-Val127 beta bridge. Phylogenetic analysis also confirmed the importance of the Tyr102 residue for enzymes from the FDH family, in which it is absolutely conserved.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from bacterium Staphylococcus aureus (SauFDH) is the most active enzyme among FDHs of this group; however, the high values of KM of the enzyme with NAD+ and formate in the standard 0.1 M phosphate buffer result in lower catalytic efficiency kcat/KM than for other FDHs. Here, we study the effect of different buffers on the catalytic properties of SauFDH. Sodium phosphate (NaPB) is used as the base buffer component and Tris, Gly and citrate (Cit) are added to NaPB to prepare double, ternary, and quaternary buffer systems with different concentrations. It is found that KM for formate does not depend on the buffer composition and concentration, while the values of kcat and $$K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}$$ increase and decrease significantly. The highest positive effect is achieved in the case of quaternary buffer NaPB-Cit-Tris-Gly. At a 0.05 M concentration of each component, kcat increases by 70% compared to one in the standard 0.1 M NaPB. At a 0.1 M of each component, improvement in both parameters, kcat and $$K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}$$ , is observed. Thermal inactivation studies in NaPB and the complex NaPB-Cit-Tris-Gly buffer showed that at component concentrations of 0.1 M and more, SauFDH’s thermal stability increased. The value of the stabilization effect depends on the ion strength but not on the type of buffer. A comparison of the X-ray structures of holo-forms of SauFDH and FDH from bacterium Pseudomonas sp.101 shows that the active site of PseFDH in complex with the substrate is totally closed, while in holo-SauFDH, amino acid residues in the active site can be accessed by water molecules and buffer components. This could be the reason of the kcat and $$K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}$$ changes in buffers of different compositions.

The 3D full-atom model of the whole-size CYP102A1 from Bacillus megaterium (cytochrome P450 BM3) has been constructed using molecular modeling methods. The structure model was constructed using crystal structures of the separate FAD-binding domain (PDB ID: 4DQK) and the complex of FMN-binding and monooxygenase domains (PDB ID: 1BVY). Modeling procedure included analysis of the domains’ surfaces to find the orientation with maximum inter-subunit contacts. The overall configuration of the obtained complex was optimized using molecular dynamics. The final full-atom structure model shows rather tight interactions between FAD- and FMN-binding domains due to 10 inter-domain hydrogen bonds and hydrophobic interactions between three pairs of amino acid residues. This 3D model can be used for structure-function studies and rational design of the enzyme as well as for construction of hybrid supramolecular structures of biocatalysts with cytochrome P450 BM3.

Phenylacetone monooxygenase (EC 1.14.13.92, PAMO) catalyzes oxidation of ketones with molecular oxygen and NADPH with the formation of esters. PAMO is a promising enzyme for biotechnological processes. In this work, we generated genetic constructs coding for PAMO from Thermobifida fusca, containing N- or C-terminal His6-tags (PAMO N and PAMO C, respectively), as well as PAMO L with the His6-tag attached to the enzyme C-terminus via a 19-a.a. spacer. All PAMO variants were expressed as catalytically active proteins in Escherichia coli BL21(DE3) cells; however, the expression level of PAMO N was 3 to 5 times higher than for the other two enzymes. The catalytic constants (kcat) of PAMO C and PAMO L were similar to that published for PAMO L produced in a different expression system; the catalytic constant for PAMO N was slightly lower (by 15%). The values of Michaelis constants with NADPH for all PAMO variants were in agreement within the published data for PAMO L (within the experimental error); however, the KM for benzylacetone was several times higher. Thermal inactivation studies and differential scanning calorimetry demonstrated that the thermal stability of PAMO N was 3 to 4 times higher compared to that of the enzymes with the C-terminal His6-tag.