Search for Inhibitors of Mycobacterium tuberculosis Transketolase in a Series of Sulfo-Substituted Compounds.

As a result of the computer screening of a library of sulfo-substituted compounds, molecules capable of binding to the active site of transketolase from Mycobacterium tuberculosis were identified. An experimental verification of the inhibitory activity of the most promising compound, STK045765, against a highly purified recombinant enzyme preparation was carried out. It was shown that the STK045765 molecule competes for the binding site of the pyrophosphate group of the thiamine diphosphate cofactor and, at a micromolar concentrations, is able to suppress the activity of mycobacterial transketolase. The discovered furansulfonate scaffold may serve as the basis for the creation of anti-tuberculosis drugs.

Specificity of Penicillin Acylases in Deprotection of N-Benzyloxycarbonyl Derivatives of Amino Acids.

Changes in the structure of the N-acyl group in N-acylated amino acid derivatives significantly affect both the recognition and activity of penicillin acylases on this series of substrates. However, penicillin acylases from both Alcaligenes faecalis and Escherichia coli are capable of removing the N-benzyloxycarbonyl protecting group in amino acid derivatives under mild conditions without the use of toxic reagents. Efficiency in using penicillin acylases in preparative organic synthesis can be improved by utilizing modern rational enzyme design methods.

Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis.

Transketolase, an enzyme of the pentose phosphate pathway, plays an important role in the functioning of mycobacteria. Using plasmid pET-19b carrying the Rv1449c gene of transketolase from Mycobacterium tuberculosis and an additional histidine tag, we isolated and purified recombinant transketolase and determined the conditions for obtaining the apoform of the protein. The Michaelis constants were evaluated for the thiamine diphosphate cofactor in the presence of magnesium and calcium ions. We found that the affinity of mycobacterial transketolase for thiamine diphosphate is by three orders of magnitude lower than that of the human enzyme. Analysis of the structural organization of the active centers of homologous enzymes showed that this difference is due to a replacement of lysine residues by less polar amino acid residues.

The role of Tyr102 residue in the functioning of bacterial NAD+-dependent formate dehydrogenase of Pseudomonas sp. 101.

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.

Analysis of Glycosyl-Enzyme Intermediate Formation in the Catalytic Mechanism of Influenza Virus Neuraminidase Using Molecular Modeling.

Using classical molecular dynamics, constant-pH molecular dynamics simulation, metadynamics, and combined quantum mechanical and molecular mechanical approach, we identified an alternative pathway of glycosyl-enzyme intermediate formation during oligosaccharide substrate conversion by the influenza H5N1 neuraminidase. The Asp151 residue located in the enzyme mobile loop plays a key role in catalysis within a wide pH range due to the formation of a network of interactions with water molecules. Considering that propagation of influenza virus takes place in the digestive tract of birds at low pH values and in the human respiratory tract at pH values close to neutral, the existence of alternative reaction pathways functioning at different medium pH can explain the dual tropism of the virus and circulation of H5N1 viral strains capable of transmission from birds to humans.

Structural Organization and Dynamic Characteristics of the Binding Site for Conformational Rearrangement Inhibitors in Hemagglutinins from H3N2 and H7N9 Influenza Viruses.

Computer models of hemagglutinins from the H3N2 and H7N9 influenza viruses were developed to study structural organization and dynamic characteristics of the binding site for the conformational rearrangement inhibitors. The metadynamics was used to map the binding site free energy and to define the volume of its most energetically favorable states. It was demonstrated by simulation of the umifenovir (Arbidol) interaction with hemagglutinin that ligand binding requires an increase in the binding site volume and deformation of its most energetically favorable state. We also identified amino acid residues directly involved in the ligand binding that determine the binding efficiency, as well as the dynamic behavior of the binding site. The revealed features of the binding site structural organization of the influenza virus hemagglutinin should be taken into account when searching for new antiviral drugs capable to modulate its functional properties.

Modeling of the Enzyme-Substrate Complexes of Human Poly(ADP-Ribose) Polymerase 1.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a key DNA repair enzyme and an important target in cancer treatment. Conventional methods of studying the reaction mechanism of PARP-1 have limitations because of the complex structure of PARP-1 substrates; however, the necessary data can be obtained by molecular modeling. In this work, a molecular dynamics model for the PARP-1 enzyme-substrate complex containing NAD+ molecule and the end of the poly(ADP-ribose) chain in the form of ADP molecule was obtained for the first time. Interactions with the active site residues have been characterized where Gly863, Lys903, Glu988 play a crucial role, and the SN1-like mechanism for the enzymatic ADP-ribosylation reaction has been proposed. Models of PARP-1 complexes with more sophisticated two-unit fragments of the growing polymer chain as well as competitive inhibitors 3-aminobenzamide and 7-methylguanine have been obtained by molecular docking.

Studying the Possibilities of Using 2-Halogen-Substituted Acetamides As Acyl Donors in Penicillin Acylase-Catalyzed Reactions.

The possibility of using amides of halogen-substituted acetic acids as acyl donors in penicillin acylase-catalyzed reactions has been investigated, and the ability of this group of compounds to inactivate enzymes in the course of the catalytic conversion has been established. The strongest inactivating effect was demonstrated by iodoacetamide and bromoacetamide. However, the negative contribution of this side activity can be minimized by decreasing the temperature, when the rate of acyl donor conversion by penicillin acylases is still high enough, but the impact of enzyme inactivation becomes less significant. The catalytic activity of penicillin acylase from Alcaligenes faecalis in the conversion of 2-haloacetamides was significantly (5-8 times) higher than that of penicillin acylase from Escherichia coli.

Isolation, Purification and Characterization of L,D-transpeptidase 2 from Mycobacterium tuberculosis.

L,D-transpeptidase 2 from Mycobacterium tuberculosis plays a key role in the formation of nonclassical 3-3 peptidoglycan cross-links in a pathogen's cell wall making it resistant to a broad range of penicillin antibiotics. The conditions of cultivation, isolation, and purification of recombinant L,D-transpeptidase 2 from M. tuberculosis have been optimized in this study. Oxidation of the free SH groups of catalytic cysteine Cys354 is an important factor causing the inactivation of the enzyme, which occurs during both the expression and storage of enzyme preparations. The biochemical characteristics of purified L,D-transpeptidase 2 and L,D-transpeptidase 2 lacking domain A were determined; the kinetic constants of enzyme-catalyzed nitrocefin transformation were evaluated.

CASBench: A Benchmarking Set of Proteins with Annotated Catalytic and Allosteric Sites in Their Structures.

In recent years, the phenomenon of allostery has witnessed growing attention driven by a fundamental interest in new ways to regulate the functional properties of proteins, as well as the prospects of using allosteric sites as targets to design novel drugs with lower toxicity due to a higher selectivity of binding and specificity of the mechanism of action. The currently available bioinformatic methods can sometimes correctly detect previously unknown ligand binding sites in protein structures. However, the development of universal and more efficient approaches requires a deeper understanding of the common and distinctive features of the structural organization of both functional (catalytic) and allosteric sites, the evolution of their amino acid sequences in respective protein families, and allosteric communication pathways. The CASBench benchmark set contains 91 entries related to enzymes with both catalytic and allosteric sites within their structures annotated based on the experimental information from the Allosteric Database, Catalytic Site Atlas, and Protein Data Bank. The obtained dataset can be used to benchmark the performance of existing computational approaches and develop/train perspective algorithms to search for new catalytic and regulatory sites, as well as to study the mechanisms of protein regulation on a large collection of allosteric enzymes. Establishing a relationship between the structure, function, and regulation is expected to improve our understanding of the mechanisms of action of enzymes and open up new prospects for discovering new drugs and designing more efficient biocatalysts. The CASBench can be operated offline on a local computer or online using built-in interactive tools at https://biokinet.belozersky.msu.ru/casbench.

Expression of glyceraldehyde-3-phosphate dehydrogenase from M. tuberculosis in E. coli. Purification and characteristics of the untagged recombinant enzyme.

The goal of the present work was to produce glyceraldehyde-3-phospate dehydrogenase from M. tuberculosis in E. coli cells in soluble and catalytically active form and to elaborate a method for the purification of the recombinant enzyme. The His-tagged recombinant enzyme (Mtb-GAPDH_His) was shown to be inactive and insoluble. The untagged enzyme (Mtb-GAPDH) was catalytically active and exhibited higher solubility. Mtb-GAPDH was purified from the cell extract using ammonium sulfate fractionation and ion-exchange chromatography. The presence of glycerol was necessary for isolation of Mtb-GAPDH, presumably, to facilitate folding of the recombinant enzyme. The yield of Mtb-GAPDH constituted 1.3 mg per 10 g of the cell biomass. The specific activity of the purified Mtb-GAPDH was 55 ±â€¯5 µmol NADH/min per mg protein (pH 9.0, 22 °C) that exceeded the activity of the previously described preparation of His-tagged recombinant GAPDH from M. tuberculosis that was co-expressed with GroEL/ES chaperone by approximately 5-fold. The results suggest that the folding of the recombinant GAPDH is hindered by the His-tag, which may result in the production of insoluble protein or in isolation of the preparation with decreased specific activity.

2,5-Diketopiperazines: A New Class of Poly(ADP-ribose)polymerase Inhibitors.

We show for the first time that natural 2,5-diketopiperazines (cyclic dipeptides) can suppress the activity of the important anticancer target poly(ADP-ribose)polymerase (PARP). Cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) can interact with the key residues of the PARP-1 active site, as demonstrated using docking and molecular dynamics simulations. One of the amide groups of cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) forms hydrogen bonds with the Gly863 residue, while the second amide group can form a hydrogen bond with the catalytic residue Glu988, and the side chain can make a hydrophobic contact with Ala898. Newly identified diketopiperazine inhibitors are promising basic structures for the design of more effective inhibitors of PARP family enzymes. The piperazine core with two chiral centers provides many opportunities for structural optimization.

Mobile Loop in the Active Site of Metallocarboxypeptidases as an Underestimated Determinant of Substrate Specificity.

It is generally accepted that the primary specificity of metallocarboxypeptidases is mainly determined by the structure of the so-called primary specificity pocket. However, the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT) with the primary specificity pocket fully reproducing the one in pancreatic carboxypeptidase B (CPB) retained the broad, mainly hydrophobic substrate specificity of the wild-type enzyme. In order to elucidate factors affecting substrate specificity of metallocarboxypeptidases and the reasons for the discrepancy with the established views, we have solved the structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N-sulfamoyl-L-phenylalanine at a resolution of 1.35 Å and compared it with the structure of similar complex formed by CPB. The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate specificity of the mutant enzyme due to the differences in the structure of the mobile loop located at the active site entrance that affects the substrate-induced conformational rearrangements of the active site.

Structure Modeling of Human Tyrosyl-DNA Phosphodiesterase 1 and Screening for Its Inhibitors.

The DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1) represents a potential molecular target for anticancer therapy. A human Tdp1 model has been constructed using the methods of quantum and molecular mechanics, taking into account the ionization states of the amino acid residues in the active site and their interactions with the substrate and competitive inhibitors. The oligonucleotide- and phosphotyrosine-binding cavities important for the inhibitor design have been identified in the enzyme's active site. The developed molecular model allowed us to uncover new Tdp1 inhibitors whose sulfo group is capable of occupying the position of the 3'-phosphate group of the substrate and forming hydrogen bonds with Lys265, Lys495, and other amino acid residues in the phosphotyrosine binding site.

Building a Full-Atom Model of L,Dtranspeptidase 2 from Mycobacterium tuberculosis for Screening New Inhibitors.

L,D-transpeptidase 2 from Mycobacterium tuberculosis plays a key role in the formation of the cell wall of a pathogen and catalyzes the cross-linking of growing peptidoglycan chains by non-classical 3-3 bonds, which causes resistance to a broad spectrum of penicillins. Molecular modeling of enzyme interactions with the N- and C-terminal tetrapeptide fragments of growing peptidoglycan chains has been performed for the first time and has allowed us to highlight the peculiarities of their binding at the formation of 3-3 cross-linkages, as well as to build a full-atom model of L,D-transpeptidase 2 for the screening and optimizing of inhibitors' structures.

Identification of New Structural Fragments for the Design of Lactate Dehydrogenase A Inhibitors.

Human lactate dehydrogenase A plays an important role in the glucose metabolism of tumor cells and constitutes an attractive target for chemotherapy. Molecular fragments able to bind in the active site of this enzyme and form hydrogen bonds with the Arg168 guanidinium group, as well as additional interactions with the loop 96-111 in the closed conformation, have been identified by virtual screening of sulfonates and experimental testing of their inhibitory effect. The sulfo group can occupy a similar position as the carboxyl group of the substrate and its structural analogs, whereas the benzothiazole group attached via a linker can be located in the coenzyme (NADH) binding site. Thus, the value of merging individual structural elements of the inhibitor by a linker was demonstrated and ways of further structural modification for the design of more effective inhibitors of lactate dehydrogenase A were established.

Inhibition of Poly(ADP-Ribose) Polymerase by Nucleic Acid Metabolite 7-Methylguanine.

The ability of 7-methylguanine, a nucleic acid metabolite, to inhibit poly(ADP-ribose)polymerase-1 (PARP-1) and poly(ADP-ribose)polymerase-2 (PARP-2) has been identified in silico and studied experimentally. The amino group at position 2 and the methyl group at position 7 were shown to be important substituents for the efficient binding of purine derivatives to PARPs. The activity of both tested enzymes, PARP-1 and PARP-2, was suppressed by 7-methylguanine with IC50 values of 150 and 50 µM, respectively. At the PARP inhibitory concentration, 7-methylguanine itself was not cytotoxic, but it was able to accelerate apoptotic death of BRCA1-deficient breast cancer cells induced by cisplatin and doxorubicin, the widely used DNA-damaging chemotherapeutic agents. 7-Methylguanine possesses attractive predictable pharmacokinetics and an adverse-effect profile and may be considered as a new additive to chemotherapeutic treatment.

Search for Human Lactate Dehydrogenase A Inhibitors Using Structure-Based Modeling.

The human lactate dehydrogenase isoform A plays an important role in the anaerobic metabolism of tumour cells and therefore constitutes an attractive target in the oncology field. Full-atom models of lactate dehydrogenase A (in complex with NADH and in the apo form) have been generated to enable structure-based design of novel inhibitors competing with pyruvate and NADH. The structural criteria for the selection of potential inhibitors were established, and virtual screening of a library of low-molecular-weight compounds was performed. A potential inhibitor, STK381370, was identified whose docking pose was stabilized through additional interactions with the loop 96-111 providing for the transition from the open to the closed conformation.

Study of Functional and Allosteric Sites in Protein Superfamilies.

The interaction of proteins (enzymes) with a variety of low-molecular-weight compounds, as well as protein-protein interactions, is the most important factor in the regulation of their functional properties. To date, research effort has routinely focused on studying ligand binding to the functional sites of proteins (active sites of enzymes), whereas the molecular mechanisms of allosteric regulation, as well as binding to other pockets and cavities in protein structures, remained poorly understood. Recent studies have shown that allostery may be an intrinsic property of virtually all proteins. Novel approaches are needed to systematically analyze the architecture and role of various binding sites and establish the relationship between structure, function, and regulation. Computational biology, bioinformatics, and molecular modeling can be used to search for new regulatory centers, characterize their structural peculiarities, as well as compare different pockets in homologous proteins, study the molecular mechanisms of allostery, and understand the communication between topologically independent binding sites in protein structures. The establishment of an evolutionary relationship between different binding centers within protein superfamilies and the discovery of new functional and allosteric (regulatory) sites using computational approaches can improve our understanding of the structure-function relationship in proteins and provide new opportunities for drug design and enzyme engineering.

Bioinformatic analysis of α/ß-hydrolase fold enzymes reveals subfamily-specific positions responsible for discrimination of amidase and lipase activities.

Superfamily of alpha-beta hydrolases is one of the largest groups of structurally related enzymes with diverse catalytic functions. Bioinformatic analysis was used to study how lipase and amidase catalytic activities are implemented into the same structural framework. Subfamily-specific positions--conserved within lipases and peptidases but different between them--that were supposed to be responsible for functional discrimination have been identified. Mutations at subfamily-specific positions were used to introduce amidase activity into Candida antarctica lipase B (CALB). Molecular modeling was implemented to evaluate influence of selected residues on binding and catalytic conversion of amide substrate by corresponding library of mutants. In silico screening was applied to select reactive enzyme-substrate complexes that satisfy knowledge-based criteria of amidase catalytic activity. Selected CALB variants with substitutions at subfamily-specific positions Gly39, Thr103, Trp104, and Leu278 were produced and showed significant improvement of experimentally measured amidase activity. Based on these results, we suggest that value of subfamily-specific positions should be further explored in order to develop a systematic tool to study structure-function relationship in enzymes and to use this information for rational enzyme engineering.