D-Amino acid oxidase (DAAO, EC 1.4.3.3) is a FAD-dependent enzyme that catalyzes the oxidative deamination of D-amino acids to produce the corresponding α-keto acids, hydrogen peroxide, and ammonium ion. High stereoselectivity toward D-enantiomers and favorable kinetic parameters make DAAO a convenient biocatalytic element for analytical applications. This review systematizes the main areas of DAAO use in bioanalysis, including clinical diagnostics, monitoring of food and biotechnological processes, and environmental surveillance. Sensor platforms and detection modes are discussed, including colorimetry, fluorimetry, chemiluminescence, electrochemistry, photoelectrochemistry, and oxygen-based detection methods. The review also addresses factors determining analytical suitability, strategies to broaden selectivity, as well as engineering approaches and structure-guided discovery of new DAAOs. Current limitations are highlighted and future prospects are outlined, such as improving enzyme stability, scaling up portable devices, and integrating biosensing with digital analytics and machine-learning algorithms.

D-Amino acid oxidase (DAAO) is an important enzyme in modern biotechnology, used for synthetic, analytical, and medical purposes. Numerous mutant forms of DAAO with different properties have been described previously. Recently, we combined six beneficial amino acid substitutions in DAAO from Trigonopsis variabilis (TvDAAO) to create the multipoint mutant TvDAAO E32R/F33D/F54S/C108F/M156L/C298N (mut-TvDAAO). Compared to the wild-type TvDAAO, the new mutant enzyme showed a fourfold higher catalytic constant in the oxidation of cephalosporin C, an eightfold higher stability against hydrogen peroxide oxidation, and a twentyfold greater thermal stability. In the present work, we demonstrate a one-step immobilization procedure of mut-TvDAAO on strong anion exchange beads Sepabeads EC-QA, yielding an immobilized biocatalyst with enhanced resistance to thermal, oxidative, alkaline, and aeration-induced stress. After immobilization, mut-TvDAAO retains approximately 40

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. In 1993, formate dehydrogenase (FDH, EC 1.2.1.2) from the methylotrophic bacterium Pseudomonas sp. 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. Since 2010, five natural NADP+-dependent FDHs have been found. In 2015-2024, three 3D structures for two natural and four 3D structures 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.

This study presents a comprehensive analysis of cysteine synthase A (CysK) from Limosilactobacillus reuteri LR1 (LreCysK), an enzyme involved in the biosynthesis of L-cysteine. This protein supports crucial cellular functions such as sulfur metabolism, antioxidant defense, detoxification, and protein synthesis. Previously, the gene encoding LreCysK was cloned, and the enzyme with His-tag on the N-terminus was obtained in active and soluble form. Here, kinetic parameters of the enzyme were determined by the previously developed high-pressure liquid chromatography (HPLC) and ninhydrin methods. It was found that LreCysK has similar KMOAS and kcat as CysKs from Escherichia coli and from the model plant Arabidopsis thaliana. The thermal stability of LreCysK was studied using differential scanning calorimetry. It was revealed that the melting point of the enzyme increases to almost 90°C when Pyridoxal-5 phosphate (PLP) is added, indicating that the stability of the enzyme complex with PLP is relatively high. Structural studies revealed that LreCysK is a dimer, and its active site is similar to those of other enzymes, but exhibits some features characteristic of lactobacilli CysKs (GISA), as well as unique residues, such as Ile50. Also, the potential biotechnological applications of LreCysK are discussed. These findings enhance our understanding of LreCysK's biochemical versatility and its potential applications in biotechnology and medicine.

Cysteine is an amino acid essential for normal functioning of living organisms. In bacteria and plants, the main mechanism of cysteine synthesis is the thiolation pathway, the second stage of which is catalyzed by either cysteine synthase A (CysK), if the substrate is inorganic sulfide, or cysteine synthase B (CysM), if the substrate is thiosulfate. The crucial role of these enzymes in cysteine synthesis makes them promising targets for antimicrobial agents and new herbicides, and well as possible components of industrial production of cysteine. In addition to their main functions, cysteine synthases show the antimicrobial and antibiofilm activities. The review discusses the physicochemical characteristics of CysK and CysM, their diversity, and potential applications in biotechnology and medicine.

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.

World-wide introduction of high throughput screening (HTS) methods in drug discovery research did not result in the increased number of novel medications on the market. We discuss novel trends in drug discovery that came from the understanding that majority of diseases are multifactorial and that one enzyme has many protein substrates. Hence, new approaches are focused on development of drugs, which (1) trigger survival pathways to return the organism to homeostatic balance, and (2) inhibit enzymes modifying histones or transcription factors not at the active site, but by displacement of protein substrates from the enzyme complexes. A good example for both approaches comes from the development of activators of antioxidant defense. We analyze and illustrate problems of commonly used in vitro HTS assays, and briefl y discuss advantages and limitations of small animal models. The novel approaches are complementary to the standard HTS and do not substitute for testing in mammals. Development of transgenic reporter mice to monitor drug effects by means of in vivo imaging is extremely promising to select proper dosage and administration regimes for full-range PK studies.

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.

Ribonucleoside hydrolase C (RihC, EC 3.2.2.1, 3.2.2.2, 3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases Rih and catalyzes the cleavage of ribonucleosides to nitrogenous bases and ribose. RihC is one of the enzymes that are synthesized by lactobacilli in response to the presence of Klebsiella. To characterize this protein from Limosilactobacillus reuteri LR1, we cloned and expressed it. The activity of the enzyme was studied towards a wide range of substrates, including ribonucleosides, deoxyribonucleosides as well as an arabinoside. It was shown that the enzyme is active only with ribonucleosides and arabinoside, with the best substrate being uridine. The thermal stability of this enzyme was studied, and its crystal structure was obtained, which demonstrated the tetrameric architecture of the enzyme and allowed to shed light on a correlation between its structure and enzymatic activity. Comprehensive comparisons of all known RihC structures, both existing crystal structures and computed model structures from various species, were made, allowing for the identification of structural motifs important for enzyme functioning.

Ribonucleoside hydrolases are enzymes that catalyze the cleavage of ribonucleosides to nitrogenous bases and ribose. These enzymes are found in many organisms: bacteria, archaea, protozoa, metazoans, yeasts, fungi and plants. Despite the simple reaction catalyzed by these enzymes, their physiological role in most organisms remains unclear. In this review, we compare the structure, kinetic parameters, physiological role, and potential applications of different types of ribonucleoside hydrolases discovered and isolated from different organisms.

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.

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.

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that addition of an Ala residue to the N-terminus reduces four-fold the rate constant of thermal inactivation compared with the addition of a Gly residue. Addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to acceleration of purification, practically does not affect kinetic parameters, but somewhat reduces thermal stability, which, however, can be restored to the level of OpaFDH_AK stability by adding 0.5 M NaCl.

As society develops, its relationship with science and influence on it becomes more and more significant. The ability to navigate the current trends in the development of society and science in particular is the most important factor in choosing new topics for scientific work and understanding the prospects for the development of scientific research. Therefore, training young professionals in this understanding (primarily in the field of natural sciences) is no less important aspect of higher education than the process of teaching fundamental and practical knowledge. This article discusses the development of relationships between society, chemistry and biotechnology (primarily applied enzymology) at different stages of human evolution. The article was written based on the materials of the introductory lecture of the section on biotechnology and applied enzymology as part of the general course “Chemical Foundations of Biological Processes”, read at the Faculty of Chemistry of Moscow State University named after M.V. Lomonosov, The features and aspects of the interaction of society, chemistry and biotechnology at different stages of the development of our world, when biotechnology has gone through the stages of development from “cave-memorable” (unconsciously natural) to “smart”, are considered. A great and important contribution to the writing of this article was made by the discussion of this problem at seminars with students of 3-6 courses of the Faculty of Chemistry of Moscow State University. The impact of changes in our society as a result of the SARS Cov-2 pandemic is also discussed separately.