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.

Activating Nrf2 with small molecules is a promising strategy for countering aging, oxidative stress, inflammation, and various disorders, including neurodegeneration. The primary regulator of Nrf2 protein stability is Keap1, a redox sensor protein and an adapter in the Cullin III ubiquitin ligase complex, which labels Nrf2 for proteasomal degradation. The canonical Nrf2 activators either chemically modify sensor thiols in Keap1 or competitively displace Nrf2 from the ubiquitin ligase complex. The latter approach is considered the most suitable for continuous administration, as non-specific chemical modifiers of Keap1 thiols also modify active thiols on other cellular proteins, causing side effects. However, when transitioning from homogeneous cell-free to cell-based assays, genuine displacement activators show a significant loss in potency by several orders of magnitude. We demonstrate that this discrepancy arises due to higher micromolar concentrations of Keap1 in cell lines. The absolute amounts of Nrf2 and Keap1 determined in brain sub-regions show more than an order of magnitude excess of Keap1 over Nrf2. A potential solution could involve targeted delivery of an alkylating agent to Keap1 to achieve the desired specificity. Transcriptomic analysis of a cell-permeable Nrf2 peptide bearing an alkylating fumarate moiety indicates selective activation of the Nrf2 genetic program, confirming the high specificity of this approach. Activation of the Nrf2-genetic program has a built-in feedback regulatory mechanism through Bach1, an Nrf2 transcriptional repressor, whose levels are elevated in age-related neurodegeneration. Thus, a benign bipartite Nrf2 activator with Bach1 inhibition properties is needed for maximal benefits. The recently developed heterocyclic carboxamide, HPPE, exhibits overlap with the Nrf2 pathway activated by the fumarate-linked Nrf2 peptide, an Nrf2 activator, as well as with zinc and tin protoporphyrins, which are inhibitors of Bach1. Therefore, HPPE presents a promising and unique combination of the two desired activities that could be further optimized to treat age-related neurodegeneration.

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.

The problem of antibiotic resistance is currently very acute. Numerous research and development of new antibacterial drugs are being carried out that could help cope with various infectious agents. One of the promising directions for the search for new antibacterial drugs is the search among the probiotic strains present in the human gastrointestinal tract. This review is devoted to characteristics of one of these probiotic strains that have been studied to date: Limosilactobacillus reuteri. The review discusses its properties, synthesis of various compounds, as well as role of this strain in modulating various systems of the human body. The review also examines key characteristics of one of the most harmful among the currently known pathogenic organisms, Klebsiella, which is significantly resistant to antibiotics existing in medical practice, and also poses a great threat of nosocomial infections. Discussion of characteristics of the two strains, which have opposite effects on human health, may help in creation of new effective antibacterial drugs without significant side effects.

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.

D-amino acid oxidase (DAAO, EC 1.2.1.2) plays an important role in the functioning of prokaryotes as well as of lower (yeast and fungi) and higher eukaryotes (mammals). DAAO genes have not yet been found in archaean genomes. D-amino acid oxidase is increasingly used in various fields, which requires the development of new variants of the enzyme with specific properties. However, even within one related group (bacteria, yeasts and fungi, mammals), DAAOs show very low homology between amino acid sequences. In particular, this fact is clearly observed in the case of DAAO from bacteria. The high variability in the primary structures of DAAO severely limits the search for new enzymes in known genomes. As a result, many (if not most) DAAO genes remain either unannotated or incorrectly annotated. We propose an approach that uses bioinformatic methods in combination with general 3D structure and active center structure analysis to confirm that the gene found encodes D-amino acid oxidase and to predict the possible type of its substrate specificity. Using a homology search, we obtained a set of candidate sequences, modelled the tertiary structure of the selected enzymes, and compared them with experimental and model structures of known DAAOs. The effectiveness of the proposed approach for discrimination of DAAOs and glycine oxidases is shown. Using this approach, new DAAO genes were found in the genomes of six strains of extremophilic bacteria, and for the first time in the world, one gene was identified in the genome of halophilic archaea. Preliminary experiments confirmed the predicted specificity of DAAO from Natronosporangium hydrolyticum ACPA39 with D-Leu and D-Phe.

Ribonucleoside hydrolase C (RihC, EC 3.2.2.1-3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases that catalyze the cleavage of both purine and pyrimidine ribonucleosides to nitrogenous bases and ribose. Its most efficient reaction is the cleavage of uridine with the highest reaction rate. The reaction cannot be detected by a simple spectrophotometric method because of the same absorption maximum for the substrate and reaction product or requires time- and labor-consuming sample preparation for ribose. Reversed-phase HPLC is currently used to register enzymatic activity, where the time of one chromatographic run takes about 10 min. Since a large number of analyses is required to measure the kinetics of an enzymatic reaction, the total time is significant. In this work, we obtained new recombinant RihC from Limosilactobacillus reuteri by gene cloning and expression in E.coli cells. We proposed a new approach for determining the enzymatic activity of the new RihC using hydrophilic interaction liquid chromatography (HILIC). The novel column was developed for this procedure providing the determination of uracil and uridine with high efficiency and retention times of 0.9 and 1.7 min, respectively. Kinetic parameters for RihC uridine cleavage were determined. The proposed approach provided significant rapidity for measurement of the enzyme kinetics being 5 times faster as compared to reversedphase HPLC.