The crystal structure of metallocarboxypeptidase T (CPT) from Thermoactinomyces vulgaris in complex with L-phenyl lactate was determined at 1.73 Å resolution. As opposed to pancreatic carboxypeptidase A, which binds one L-phenyl lactate molecule, the ligand in the complex with CPT occupies simultaneously the S1 and S' subsites of the active site. This leads to conformational changes, which differ from those caused by the alternating occupation of the S1 and S1' subsites by tert-butyloxycarbonyl-L-leucine (BOC-leucine) and benzylsuccinic acid. These changes concern the residues E277, E59, L254, G192, S127, and Y218 and are up to 0.77 Å. A conclusion was drawn about the possible role of the residue E59 in the substrate recognition and catalysis by carboxypeptidase Т.

A highly effective producer strain Escherichia coli C3030/pET23d + - Ec HGPRT, allowing production of recombinant hypoxanthine‒guanine phosphoribosyltransferase from E. coli ( Ec HGPRT) in a soluble form, has been created. A method for isolating and purifying the recombinant protein has been developed. The specific activity against the natural substrate and pyrazine-2-carboxamide derivatives has been determined. Crystals of the Ec HGPRT complexes with 3-hydroxypyrazine-2-carboxamide (T-1105) and 6-fluoro-3-hydroxypyrazine-2-carboxamide (T-705), suitable for X-ray diffraction analysis, have been grown by capillary counter diffusion. X-ray diffraction sets with a resolution of up to 2.4 and 2.5 Å have been collected at the ESRF synchrotron (France, station ID23-1) at a temperature of 100 K. The crystals belong to the sp. gr. P 3(1)21; the independent part of the cell contains two enzyme molecules.

The high-producing strain С3029/pGro7/pERDerp3 for the house dust mite allergen Der p 3 from Dermatophagoides pteronyssinus , expressing the recombinant protein in Escherichia coli in the soluble form, was constructed. A procedure was developed for the purification of the recombinant allergen. Crystals of the recombinant protein Der p 3 suitable for X-ray diffraction analysis were grown by the vapor-diffusion method. The X-ray diffraction data set was collected to 2.25 Å resolution at the European Synchrotron Radiation Facility (ESRF, France, ID23-1 beamline) at 100 K. The crystals belong to sp. gr. С 121 and contain two enzyme molecules per asymmetric unit.

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the β-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the β-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

Wild-type L-asparaginase from Wolinella succinogenes and its double mutant with V23Q and K24T substitutions were studied by molecular dynamics simulations. The molecular dynamics trajectories were analyzed. It was shown that the mobility of atoms in the mutant protein is much higher than that in the wild-type protein. The structural elements of the protein, the mobility of which is most strongly influenced by mutations, were revealed. The obtained data provide an explanation of changes in the substrate specificity of the mutant L-asparaginase from Wolinella succinogenes.

Abstract Two recombinant purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27 encoded by genes TT_C1070 (TthPNPI) and TT_C0194 (TthPNPII) were purified and characterized. The comparative analysis of their sequences, molecular weight, enzymes specificity and kinetics of the catalyzed reaction were realized. As a result, it was determined that the TthPNPI is specific to guanosine while the TthPNPII to adenosine. According to the results of the size exclusion chromatography and SAXS study both enzymes are hexameric molecules. Based on the sequence alignment with homologous purine nucleoside phosphorylases (PNPs), Asn was identified as a purine base recognizing residue in the active site of TthPNPI and Asp in TthPNPII. The three-dimensional structure of TthPNPII was solved at 2.5 Å resolution by molecular replacement method using crystals grown in microgravity. Position of phosphate in the active site cavity is located. The possible arrangement of adenosine and guanosine in TthPNPII active site cavity is considered using superposition with the structures of homologous trimeric and hexameric PNPs complexed with corresponding substrates. The peculiarities of oligomeric structure of TthPNPII in comparison with homologous PNPs are described. It is shown that two trimeric molecules of TthPNPII in the asymmetric part of the unit cell are connected by three two-fold axis into a hexamer with 32-point symmetry. This type of hexameric structure of PNP is found for the first time. The interface area between the subunits in trimeric molecule and between the trimers in TthPNPII hexamer is described. Communicated by Ramaswamy H. Sarma.

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states—closed and open—in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Carboxypeptidase T (CPT; EC 3.4.17.18) from Thermoactinomyces vulgaris is a distant homolog of the highly specific pancreatic carboxypeptidase B; but has a broad substrate specificity; the source of which remains unclear. A previous study of the structural bases of the substrate specificity of CPT using stable sulfamoyl analogs of the transition state of the elimination of leucine; phenylalanine; arginine; and glutamic acid; showed that the binding of the C-terminal residue of the substrate to the primary selectivity pocket of CPT leads to a change in the distance between Zn2+ and the sulfur atom. This value is related to the efficiency of catalysis of the corresponding substrate or the inhibition constant of the corresponding stable analog of the transition state. In this work; we obtained crystallographic and kinetic data of the complex of CPT with N-sulfamoyl-L-valine; confirming the effect of the binding of the ligand’s side group by the primary specificity pocket of CPT on the structure of the catalytic center; which can explain the unusual substrate specificity of CPT.

A covalent serine protease inhibitor—Na-p-Tosyl-Lysyl Chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues.

We report the spatial structure of phosphoribosyl pyrophosphate synthetase 2 from the thermophilic bacterium Thermus thermophilus HB27 (TthPRPPS2) obtained at a 1.85 Å resolution using a diffraction set collected from rhombohedral crystals (space group R32-h), grown with lithium sulfate as a precipitant. This crystal structure was compared with the structure of TthPRPPS2, previously obtained at a 2.2 Å resolution using diffraction sets from the tetragonal crystals (space group P41212), grown with ammonium sulfate as a precipitant. The comparison of these structures allows the study of the differences between protein molecules in both crystalline structures, as well as the packaging of enzyme molecules in crystals of both spatial groups. Our results may contribute to the research of the structural basis of catalytic activity and substrate specificity of this enzyme.