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A M Golubev

Publications and source records attributed to A M Golubev.

At least 19 recordsLinked to original sources
PubMed2005

Getting the most out of X-ray home sources.

The structures of a 14 kDa phospholipase, an 18 kDa proteinase inhibitor and a novel glycoside hydrolase with molecular weight 60 kDa were solved using the SAD technique and the effects of the amount of anomalous signal, completeness and redundancy of data on heavy-atom substructure determination, phasing and model building were analyzed. All diffraction data sets were collected on a Cu-anode X-ray home source. The structure of the phospholipase was obtained using the anomalous scattering contribution from its 16 S atoms. Three-dimensional models for the other two macromolecules were obtained using the anomalous contribution of I atoms rapidly incorporated into the crystal through the quick cryo-soaking method of derivatization. These results were used to discuss the application of sulfur- and iodine-SAD approaches in combination with X-ray home sources for high-throughput protein crystal structure solution. The estimates of the anomalous signal from S atoms in the gene products of four genomes are given and the prospects for increasing the anomalous contribution using longer wavelengths (e.g. from a chromium home source) and quick cryo-soaking derivatization are discussed. The possibility of rapidly preparing tangible home-source isomorphous derivatives suggests that this approach might become a valuable tool in the future of post-genomic projects.

Aspergillus↗
PubMed2004

Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition.

Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked beta-d-fructofuranose residues in inulin, levan and sucrose releasing beta-d-fructose. We present the X-ray structure at 1.55A resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed beta-propeller fold and the C-terminal domain folded into a beta-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (beta-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87A, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.

Amino Acid Motifs↗
PubMed2004

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose.

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.

Amino Acid Sequence↗
PubMed2004

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

Binding Sites↗
PubMed2000

Purification, crystallization and preliminary X-ray study of beta-xylosidase from Trichoderma reesei.

An extracellular multifunctional beta-xylosidase was purified from a culture of the fungus Trichoderma reesei. The active 95 +/- 5 kDa enzyme has been crystallized from sodium acetate buffer using PEG as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.75, b = 98.54, c = 227.25 A, and diffract beyond 2.7 A resolution. X-ray data were collected from frozen crystals on a synchrotron source.

Crystallization↗
PubMed2000

[Intraperitoneal perfusion of perftoran in the treatment of patients with diffuse purulent peritonitis].

For higher efficacy of general purulent peritonitis treatment, intraperitoneal perfusion of Perftoran emulsion was proposed. The drug was administered as a single dose of 100-150 ml before wound suturing. In 48 hours an increase of phagocytizing cells fraction in peritoneal exudate and their higher functional activity was revealed: the phagocytic activity and phagocytic index was 2 times higher than in the control group, complete phagocytosis index--1.5 times higher. Significant reduction of microbial contamination in abdominal cavity was also revealed: in 48 hours there were less aerobic bacteria than in the control group, there were no anaerobic bacteria, or anaerobes were detected in a minimum quantity. Local use of Perftoran allowed to decrease the rate of repeated peritoneal sanitation by 44.4% and to decrease mortality by 5.2%.

Bacteria↗
PubMed1999

Purification, crystallization and preliminary crystallographic study of a Kunitz-type trypsin inhibitor from Delonix regia seeds.

The Kunitz-type trypsin inhibitor from seeds of Flamboyant (Delonix regia) has been purified to homogeneity and plate-like crystals suitable for X-ray analysis have been grown by the hanging-drop method using PEG 6000 as a precipitant. The crystals belong to space group P2(1)2(1)2(1) with unit-cell parameters a = 32.15, b = 69.39, c = 72.54 A. X-ray diffraction data have been collected to 2.95 A resolution. The structure has been solved by molecular replacement using the known structures of trypsin inhibitors from Erythrina caffra seeds (PDB code 1tie) and from soya beans (Glycine max; PDB code 1ba7) as search models.

Crystallization↗
PubMed1999

Crystallization and preliminary crystal analysis of yeast hexokinase PI and PII.

Hexokinase is the prime enzyme of the Embden-Meyerhof pathway and is responsible for the first stage of energy conversion. It catalyzes the transfer of a phosphate to glucose to form glucose-6-phosphate. Yeast hexokinase PII is also known to play an important role in glucose signal transduction. Crystals of yeast hexokinase isoforms PI and PII were obtained by vapour-diffusion techniques using the hanging-drop method. Isoform PI crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 62.12, b = 78.87, c = 144.74 A. Unit-cell parameters for isoform PII crystals are a = b = 142.81, c = 58.46 A and the space group is I4. Synchrotron diffraction data have been collected to 2.2 A resolution from the isoform PII crystal, whereas isoform PI diffracted to 3.1 A.

Crystallization↗
PubMed1998

Crystallization and preliminary diffraction data of neurotoxin Ts-gamma from the venom of the scorpion Tityus serrulatus.

Scorpion neurotoxin Ts-gamma was isolated from Tityus serrulatus venom and purified to apparent homogeneity by ion-exchange HPLC. Crystals of the toxin were grown using polyethylene glycol 6000 as precipitant and were found to belong to the monoclinic space group P21 with cell parameters a = 22.20, b = 36.90, c = 31.57 A, beta = 100.85 degrees. The crystals diffract beyond 1.73 A resolution at a synchrotron beamline, being notably stable during X-ray exposure. The structure has been solved by molecular replacement using the very high resolution structure of Sahara scorpion Androctonus australis Hector (PDB code 1AHO) as a search model.

Animals↗
PubMed1998

alpha-Mannosidase from Trichoderma reesei participates in the postsecretory deglycosylation of glycoproteins.

The 160 kDa alpha-mannosidase (E.C. 3.2.1.24) isolated from culture filtrate of Trichoderma reesei has wide aglycon specificity but cleaves the alpha1 --> 2 and alpha1 --> 3 mannosidic bonds with higher rate than alpha1 --> 6 bond and slowly hydrolyses yeast mannan and 1,6-alpha-mannan. The specific activity of the enzyme and rate constant in the reaction with p-nitrophenyl-alpha-D-mannopyranoside were 0.15 U/mg and 1.62 x 10(-4) microM/min/microg, respectively, at optimal pH 6.5. We have found that in vitro enzyme is able to cleave off 30% of total alpha-mannopyranosyl residues from N- and O-linked glycans of secreted glycoproteins. The activity of the alpha-mannosidase toward glycoproteins in vivo was studied comparing the structures of O- and N-linked glycans of glycoproteins isolated from the cultures growing with and without 1-deoxymannojirimycin, an inhibitor of alpha-mannosidases. Difference in structures of these glycans may be explained by postsecretory deglycosylation catalysed by the alpha-mannosidase.

1-Deoxynojirimycin↗
PubMed1998

Tryptophan residues in alpha-galactosidase from Trichoderma reesei.

Tryptophan residues in alpha-galactosidase were modified with bromosuccinimide. The fact that galactose, a specific inhibitor of alpha-galactosidase, does not prevent this modification demonstrates that tryptophan residues are not located in galactose binding sites. Analysis of the inactivation kinetics revealed two groups of Trp residues (8.5 and 7.5 residues) with different accessibility for N-bromosuccinimide. We studied specific quenching of alpha-galactosidase fluorescence resulting from modification of an sulfhydryl group in the active site of the enzyme with Hg2+ and Ag+ ions. The specific quenching is due to conformational changes of the enzyme. Forster's radii were determined for various protein--chromophore complexes. Dynamic quenching of alpha-galactosidase fluorescence was investigated. To describe abnormal dynamic quenching in alpha-galactosidase, a modification of the Stern--Volmer equation is suggested.

Bromosuccinimide↗
PubMed1997

Transglycosylation activity of alpha-D-galactosidase from Trichoderma reesei. An investigation of the active site.

The transglycosylation reaction catalyzed by alpha-D-galactosidase from the mycelial fungus Trichoderma reesei was studied using p-nitrophenyl alpha-D-galactopyranoside (PNPG). An aliphatic alcohol or the substrate itself can be an acceptor of the galactose residue in this reaction. The transglycosylation products were identified as alkyl galactosides in the case of alcohols or as galactobioside and galactotrioside in the case of PNPG. The transglycosylation rates follow a first-order equation with respect to the alcohol concentrations except for methanol. Affinities of some substrates were estimated from their Ki values in the reaction of the enzyme with PNPG. Transglycosylation of the substrate suggests a model for the enzyme active center. It is proposed that the active center includes two galactose-binding sites and a hydrophobic site.

Binding Sites↗
PubMed1997

The carbohydrate moiety of alpha-galactosidase from Trichoderma reesei.

Alpha-galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by beta-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: alpha1-6 D-mannopyranosyl-alpha-D-glycopyranoside and alpha1-6 D-glucopyranosyl-alpha-D-galactopyranoside and one trimer: alpha-D-glucopyranosyl-alpha1-2 D-mannopyranosyl-alpha1-6 D-galactopyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo-beta-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo-beta-N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of alpha-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of alpha-galactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described.

Amino Acid Sequence↗
PubMed1996

[The biomicroscopic evaluation of the blood microcirculatory bed in the mesentery of the small intestine during experimental perftoran infusion under normovolemia].

Dynamics of biomicroscopic state of hemomicrocirculatory bed (HMCB) of small intestinal mesentery was studied in go both sexes albino outbred rats in conditions of intravenous administration of 1.0 ml perfuoram (PF) with simultaneous control of the experiment by intravenous isotonic solution administration. Biomicroscopy was performed 30 minutes, 3, 6 and 24 hours later. 30 minutes after the PF administration bloodflow reduction in afferent vessels, elongation of construction period of resistant vessels and 18% decrease of vessels were demonstrated. Precapillary diameter decreased on 23% with respect to control with isotonic solution. Tortuosity of precapillaries and decrease of functioning capillaries number. 3 hours after the PF infusion blood flow turned more slow, signs of adhesion between leukocytes and postcapillary venules lumenal surface appeared and perivascular infiltrate had formed. Diameter of alducent vessels reduced in comparison with control and went lugher against the 30-minutes research stage. On the whole bloodflow in resistant vessels turns homogeneous. 6 hours after PF infusion homogeneous bloodflow could be seen. Number of arteriolar vasomotions grew up to control and made 14 in a minute. Venular diameter was close to control. Biomicroscopy of HMCB 24 hours after infusion did not reveal essential differences compared to with isotonic solution administered.

Animals↗
PubMed1995

Role of methionine in the active site of alpha-galactosidase from Trichoderma reesei.

alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide.

Amino Acids↗