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1.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 5): 541-52, 2012 May.
Article in English | MEDLINE | ID: mdl-22525752

ABSTRACT

The analysis reported here describes detailed structural studies of endothiapepsin (the aspartic proteinase from Endothia parasitica), with and without bound inhibitors, and human pepsin 3b. Comparison of multiple crystal structures of members of the aspartic proteinase family has revealed small but significant differences in domain orientation in different crystal forms. In this paper, it is shown that these differences in domain orientation do not necessarily correlate with the presence or absence of bound inhibitors, but appear to stem at least partly from crystal contacts mediated by sulfate ions. However, since the same inherent flexibility of the structure is observed for other enzymes in this family such as human pepsin, the native structure of which is also reported here, the observed domain movements may well have implications for the mechanism of catalysis.


Subject(s)
Aspartic Acid Proteases/chemistry , Ascomycota/enzymology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Proteases/antagonists & inhibitors , Crystallography, X-Ray , Humans , Models, Molecular , Pepsin A/antagonists & inhibitors , Pepsin A/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Conformation , Protein Structure, Tertiary
2.
J Biol Chem ; 276(29): 27498-510, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11342548

ABSTRACT

In the ferredoxin-NADP(+) reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investigated further the role of hydrophobic interactions in complex stabilization and ET between these proteins by replacing three hydrophobic residues, Leu-76, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from NADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chains. In contrast, only subtle changes are observed upon conservative replacement. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of these mutants. The introduction of bulky aromatic side chains appears to produce rearrangements of the side chains at the FNR/Fd interaction surface. Thus, subtle changes in the hydrophobic patch influence the rates of ET to and from Fd by altering the binding constants and the FAD redox potentials, indicating that these residues are especially important in the binding and orientation of Fd for efficient ET. These results are consistent with the structure reported for the Anabaena FNR.Fd complex.


Subject(s)
Anabaena/enzymology , Ferredoxin-NADP Reductase/metabolism , Ferredoxins/metabolism , Flavin-Adenine Dinucleotide/physiology , Multigene Family , Amino Acid Sequence , Electron Transport , Ferredoxin-NADP Reductase/chemistry , Flavin-Adenine Dinucleotide/chemistry , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Conformation , Sequence Homology, Amino Acid
3.
J Biol Chem ; 276(15): 11902-12, 2001 Apr 13.
Article in English | MEDLINE | ID: mdl-11152461

ABSTRACT

On the basis of sequence and three-dimensional structure comparison between Anabaena PCC7119 ferredoxin-NADP(+) reductase (FNR) and other reductases from its structurally related family that bind either NADP(+)/H or NAD(+)/H, a set of amino acid residues that might determine the FNR coenzyme specificity can be assigned. These residues include Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235. Systematic replacement of these amino acids was done to identify which of them are the main determinants of coenzyme specificity. Our data indicate that all of the residues interacting with the 2'-phosphate of NADP(+)/H in Anabaena FNR are not involved to the same extent in determining coenzyme specificity and affinity. Thus, it is found that Ser-223 and Tyr-235 are important for determining NADP(+)/H specificity and orientation with respect to the protein, whereas Arg-224 and Arg-233 provide only secondary interactions in Anabaena FNR. The analysis of the T155G FNR form also indicates that the determinants of coenzyme specificity are not only situated in the 2'-phosphate NADP(+)/H interacting region but that other regions of the protein must be involved. These regions, although not interacting directly with the coenzyme, must produce specific structural arrangements of the backbone chain that determine coenzyme specificity. The loop formed by residues 261-268 in Anabaena FNR must be one of these regions.


Subject(s)
Coenzymes/metabolism , Ferredoxin-NADP Reductase/metabolism , Amino Acid Sequence , Circular Dichroism , Ferredoxin-NADP Reductase/chemistry , Ferredoxin-NADP Reductase/genetics , Kinetics , Models, Molecular , Molecular Probes , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Sequence Homology, Amino Acid , Spectrophotometry, Ultraviolet , Substrate Specificity
4.
J Mol Biol ; 300(2): 363-75, 2000 Jul 07.
Article in English | MEDLINE | ID: mdl-10873471

ABSTRACT

Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 A resolution, complexed with a methionine analogue: l-2-amino-4-methoxy-cis-but-3-enoic acid (l-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The l-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg(2+), which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design.


Subject(s)
Liver/enzymology , Methionine Adenosyltransferase/chemistry , Methionine Adenosyltransferase/metabolism , Methionine/metabolism , Acid Anhydride Hydrolases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Substitution/genetics , Animals , Binding Sites , Crystallography, X-Ray , Cysteine/metabolism , Dimerization , Disulfides/chemistry , Disulfides/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Methionine/analogs & derivatives , Methionine Adenosyltransferase/antagonists & inhibitors , Methionine Adenosyltransferase/genetics , Models, Molecular , Mutation/genetics , Oxidation-Reduction , Protein Binding , Protein Folding , Protein Renaturation , Protein Structure, Quaternary , Protein Structure, Tertiary , Rats
5.
J Biol Chem ; 275(18): 13708-12, 2000 May 05.
Article in English | MEDLINE | ID: mdl-10788490

ABSTRACT

The beta-glucosidase encoded by the bglA gene from Paenibacillus polymyxa has a half-life time of 15 min at 35 degrees C and no detectable activity at 55 degrees C. We have isolated random mutations that enhance the thermoresistance of the enzyme. Following a directed evolution strategy, we have combined some of the isolated mutations to obtain a beta-glucosidase with a half-life of 12 min at 65 degrees C, in the range of resistance of thermophilic enzymes. No significant alteration of the kinetic parameters of the enzyme was observed. One of the mutants isolated in the screening for thermoresistant beta-glucosidase had the same resistance to denaturation as the wild type. This mutation caused the accumulation of enzyme in E. coli, probably due to its lower turnover. The structural changes responsible for the properties of the mutant enzymes have been analyzed. The putative causes increasing thermoresistance are as follows: the formation of an extra salt bridge, the replacement of an Asn residue exposed to the solvent, stabilization of the hydrophobic core, and stabilization of the quaternary structure of the protein.


Subject(s)
Bacillus/physiology , beta-Glucosidase/physiology , Adaptation, Physiological , Evolution, Molecular , Mutation , Protein Conformation , Structure-Activity Relationship , Temperature , beta-Glucosidase/chemistry
6.
Proteins ; 38(1): 60-9, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10651039

ABSTRACT

The three-dimensional crystal structure of the Glu301Ala site-directed mutant of ferredoxin-NADP+ reductase from Anabaena PCC 7119 has been determined at 1.8A resolution by x-ray diffraction. The overall folding of the Glu301Ala FNR mutant shows no significant differences with respect to that of the wild-type enzyme. However, interesting conformational changes are detected in the side chain of another glutamate residue, Glu139, which now points towards the FAD cofactor in the active center cavity. The new conformation of the Glu139 side chain is stabilized by a network of five hydrogen bonds to several water molecules, which seem to hold the carboxylate side chain in a rather fixed position. This interacting network connects the Glu139 side chain to the Ser80 side chain through a series of three water molecules. These observations are discussed in terms of the reactivity of Glu301Ala ferredoxin-NADP+ reductase towards its substrates, and the role of Glu301 in the catalysis is re-examined. Moreover, a structural explanation of the different reoxidation properties of this mutant is given on the basis of the reported structure by modeling the hypothetical flavin C(4a)-hydroperoxide intermediate. The model shows that the distal oxygen of the peroxide anion could be in an appropriate situation to act as the proton donor in the reoxidation process.


Subject(s)
Anabaena/enzymology , Ferredoxin-NADP Reductase/chemistry , Anabaena/genetics , Catalytic Domain/genetics , Computer Simulation , Crystallography, X-Ray , Ferredoxin-NADP Reductase/genetics , Models, Molecular , Mutagenesis, Site-Directed , Point Mutation , Protein Conformation , Solvents , Spinacia oleracea/enzymology , Spinacia oleracea/genetics , Water/chemistry
7.
Proteins ; 33(4): 567-76, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9849940

ABSTRACT

The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions. beta-glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering. This is a well-characterized enzyme, belonging to glycosyl hydrolase family 1. Its natural substrate is cellobiose, but is also active against various artificial substrates. In its native state has an octameric structure. Its subunit conserves the general (alpha/beta)8 barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel. Using random-mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes. The crystal structure of this mutant has been determined by X-ray diffraction and compared with the native structure. The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N-terminus of helix alpha2, to Asp28, located in one of the loops surrounding the active-site cavity. The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure. Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed. However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other. These results show that far-reaching effects can be achieved by a single amino acid replacement within the protein structure. Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions.


Subject(s)
Enzyme Stability , Protein Structure, Secondary , beta-Glucosidase/chemistry , Amino Acid Sequence , Bacillus/enzymology , Base Sequence , Circular Dichroism , Crystallography, X-Ray , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis , Point Mutation , Protein Denaturation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Thermodynamics , beta-Glucosidase/genetics
8.
J Mol Biol ; 275(3): 491-502, 1998 Jan 23.
Article in English | MEDLINE | ID: mdl-9466926

ABSTRACT

Family 1 glycosyl hydrolases are a very relevant group of enzymes because of the diversity of biological roles in which they are involved, and their generalized occurrence in all sorts of living organisms. The biological plasticity of these enzymes is a consequence of the variety of beta-glycosidic substrates that they can hydrolyze: disaccharides such as cellobiose and lactose, phosphorylated disaccharides, cyanogenic glycosides, etc. The crystal structure of BglA, a member of the family, has been determined in the native state and complexed with gluconate ligand, at 2.4 A and 2.3 A resolution, respectively. The subunits of the octameric enzyme display the (alpha/beta)8 barrel structural fold previously reported for other family 1 enzymes. However, significant structural differences have been encountered in the loops surrounding the active-center cavity. These differences make a wide and extended cavity in BglA, which seems to be able to accommodate substrates longer than cellobiose, its natural substrate. Furthermore, a third sub-site is encountered, which might have some connection with the transglycosylating activity associated to this enzyme and its certain activity against beta-1,4 oligosaccharides composed of more than two units of glucose. The particular geometry of the cavity which contains the active center of BglA must therefore account for both, hydrolytic and transglycosylating activities. A potent and well known inhibitor of different glycosidases, D-glucono-1,5-lactone, was used in an attempt to define interactions of the substrate with specific protein residues. Although the lactone has transformed into gluconate under crystallizing conditions, the open species still binds the enzyme, the conformation of its chain mimicking the true inhibitor. From the analysis of the enzyme-ligand hydrogen bonding interactions, a detailed picture of the active center can be drawn, for a family 1 enzyme. In this way, Gln20, His121, Tyr296, Glu405 and Trp406 are identified as determinant residues in the recognition of the substrate. In particular, two bidentate hydrogen bonds made by Gln20 and Glu405, could conform the structural explanation for the ability of most members of the family for displaying both, glucosidase and galactosidase activity.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Glucosidases/chemistry , Glucosidases/metabolism , Glycoside Hydrolases/metabolism , Amino Acid Sequence , Bacillus/enzymology , Binding Sites , Catalysis , Crystallography, X-Ray , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Folding , Sequence Homology, Amino Acid
9.
Biochemistry ; 37(51): 17680-91, 1998 Dec 22.
Article in English | MEDLINE | ID: mdl-9922134

ABSTRACT

Previous studies and the crystal structure of Anabaena PCC 7119 FNR suggest that the side chains of Arg100 and Arg264 may be directly involved in the proper NADP+/NADPH orientation for an efficient electron-transfer reaction. Protein engineering on Arg100 and Arg264 from Anabaena PCC 7119 FNR has been carried out to investigate their roles in complex formation and electron transfer to NADP+ and to ferredoxin/flavodoxin. Arg100 has been replaced with an alanine, which removes the positive charge, the long side chain, as well as the ability to form hydrogen bonds, while a charge reversal mutation has been made at Arg264 by replacing it with a glutamic acid. Results with various spectroscopic techniques indicate that the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Both mutants have been kinetically characterized by steady-state as well as fast transient kinetic techniques, and the three-dimensional structure of Arg264Glu FNR has been solved. The results reported herein reveal important conceptual information about the interaction of FNR with its substrates. A critical role is confirmed for the long, positively charged side chain of Arg100. Studies on the Arg264Glu FNR mutant demonstrate that the Arg264 side chain is not critical for the nicotinamide orientation or for nicotinamide interaction with the isoalloxazine FAD moiety. However, this mutant showed altered behavior in its interaction and electron transfer with its protein partners, ferredoxin and flavodoxin.


Subject(s)
Anabaena/enzymology , Arginine/metabolism , Ferredoxin-NADP Reductase/chemistry , Ferredoxin-NADP Reductase/metabolism , NADP/metabolism , Alanine/genetics , Anabaena/genetics , Arginine/genetics , Circular Dichroism , Computer Simulation , Crystallography, X-Ray , Electron Transport , Escherichia coli/enzymology , Escherichia coli/genetics , Ferredoxin-NADP Reductase/genetics , Glutamic Acid/genetics , Kinetics , Models, Molecular , Oxidation-Reduction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Spectrophotometry , Substrate Specificity/genetics
10.
J Org Chem ; 63(21): 7157-7161, 1998 Oct 16.
Article in English | MEDLINE | ID: mdl-11672354

ABSTRACT

Lithium dienolate of 3-butenoic methyl ester was reacted with enantiomerically pure N-arylsulfinyl phenylimines (1 and 2) under different conditions. The reactions were completely regioselective-the C-C coupling occurs at the 2-position of dienolate-and highly stereoselective at the iminic carbon. In the presence of different Lewis acids (ZnCl(2), ZnBr(2), and ScTf(3)) mixtures of two alpha-vinyl, beta-arylsulfinylamino esters (epimers at C-alpha) were obtained, being the stereoselectivity depending on the nature of the aryl sulfinyl moiety and the Lewis acid used. Desulfinylation of these mixtures followed by isomerization of the double bond with Na(2)CO(3) allowed the synthesis of the optically pure (E)-alpha-ethylidene-beta-amino ester 10 in quite high overall yield. The addition of the lithium dienolate to sulfinylimines in the absence of the Lewis catalysts yielded mixtures containing important amounts of the optically pure N-arylsulfinyl alpha-ethylidene-beta-amino esters, which became the exclusive product of the reaction when N-2-methoxynaphthylsulfinyl phenylimine 2 was used as starting product.

11.
Acta Crystallogr D Biol Crystallogr ; 53(Pt 3): 321-6, 1997 May 01.
Article in English | MEDLINE | ID: mdl-15299936

ABSTRACT

C-phycocyanin and allophycocyanin from the green alga Spirulina platensis were isolated and crystallized by gel-acupuncture techniques. A novel two-step chromatographic procedure was used for purification. Blue hexagonal crystals were obtained by diffusing magnesium chloride into the protein solution for a week, followed by diffusion of PEG 6000 in order to complete the reduction of the solubility of the protein in the capillary tube used as a growth cell. In the case of allophycocyanin, crystals with a size of 0.4 x 0.3 x 0.3 mm were characterized by X-ray diffraction. They belong to space group P6(3)22 with unit-cell parameters a = b = 102.04, c = 131.22 A. The crystals of C-phycocyanin belong to either space group P6 or P6(3) with unit-cell constants a = b = 182.38, c = 60.87 A, alpha = beta = 90, gamma = 120 degrees. The crystals diffract beyond 2.4 and 2.5 A resolution, respectively, using a rotating anode as an X-ray source.

12.
FEBS Lett ; 405(1): 114-8, 1997 Mar 17.
Article in English | MEDLINE | ID: mdl-9094437

ABSTRACT

Canavalia brasiliensis lectin was isolated from the seeds of a Brazilian autochthonous Leguminosae plant. Despite extensive amino acid sequence similarity with Concanavalin A, C. brasiliensis lectin exerts in vitro and in vivo cellular effects that are markedly different from those displayed by Concanavalin A. We have solved the crystal structure of the C. brasiliensis lectin at 3.0 A resolution. The three-dimensional structure of the lectin monomer can be superimposed onto that of Concanavalin A with a root-mean-square deviation for all C alpha atoms of 0.65 A. However, this parameter is 0.84 and 1.62 A when the C. brasiliensis lectin dimer and tetramer, respectively, are compared with the same structures of Concanavalin A. We suggest that these differences in quaternary structure may account for the different biological properties of these two highly related Leguminosae lectins.


Subject(s)
Concanavalin A/chemistry , Lectins/chemistry , Crystallography, X-Ray , Fabaceae , Lectins/physiology , Models, Molecular , Plant Lectins , Plants, Medicinal , Protein Conformation , Structure-Activity Relationship
13.
J Med Chem ; 38(15): 2830-41, 1995 Jul 21.
Article in English | MEDLINE | ID: mdl-7636844

ABSTRACT

The synthesis and pharmacological activities of the four stereoisomers of methyl tetrahydrofuran-2-ylmethyl 2,6-dimethyl-4-(2'-nitrophenyl)-1,4-dihydropyridine-3,5- dicarboxylate(furnidipine) are reported. The four isomers were synthesized by a modified Hantzsch synthesis by reaction of (-)- or (+)-tetrahydrofuran-2-ylmethyl 3-aminocrotonate and methyl 2-[(2'-nitrophenyl)methylene]acetoacetate or, alternatively, by reaction of (-)- or (+)-tetrahydrofuran-2-ylmethyl 2-[(2'-nitrophenyl)methylene]acetoacetate and methyl 3-aminocrotonate. The 1:1 diastereomeric mixtures thus obtained were separated by chromatography, using poly(D-phenylglycine) as the chiral stationary phase. The enantiomeric purity of the stereoisomers was determined by a high-performance liquid chromatography-chiral stationary phase technique (HPLC-CSP). Attempts to obtain crystals of a single stereoisomer failed in different solvents, while methanol crystallization of the product obtained from (+/-)-tetrahydrofuran-2-ylmethyl 2-[(2'-nitrophenyl)methylene]acetoacetate and methyl 3-aminocrotonate yielded good-quality crystals of the most insoluble racemate which proved to be a mixture of the (SS)/(RR) enantiomers by X-ray crystallography. Conformational analysis of the stereoisomers, assuming rotation of the aryl substituent and ester groups, shows small energy differences (about 4 kcal.mol-1) between the most and the least favorable conformations. Binding studies were performed using [3H]isradipine as a reference ligand. The results showed stereospecificity of the furnidipine isomers in brain, ileum, and cardiac tissues, the (SS)- and (SR)-isomers clearly being more potent than their (RR)- and (RS)-enantiomers. The (SS)- and (SR)-isomers were also more selective on cerebral tissue when compared with ileal and cardiac preparations.


Subject(s)
Calcium Channel Blockers/chemistry , Calcium Channel Blockers/pharmacology , Dihydropyridines/chemistry , Dihydropyridines/pharmacology , Animals , Binding, Competitive , Brain/drug effects , Brain/metabolism , Calcium Channel Blockers/chemical synthesis , Chemical Phenomena , Chemistry, Physical , Crystallography, X-Ray , Dihydropyridines/chemical synthesis , Drug Evaluation, Preclinical , Guinea Pigs , Heart/drug effects , In Vitro Techniques , Isradipine/metabolism , Kinetics , Molecular Conformation , Molecular Structure , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/drug effects , Myocardial Contraction/drug effects , Myocardium/metabolism , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity Relationship , Tritium
14.
J Pharm Sci ; 84(1): 101-6, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7714729

ABSTRACT

A series of tropane-3-spiro-4'(5')-imidazolines was synthesized and studied by 1H and 13C NMR spectroscopy, and the crystal structure of 2'-(1H-indol-3-yl)tropane-3-spiro-4'(5')-imidazoline hydrochloride 5(6)f was determined by X-ray diffraction. In CD3OD solution, compounds 5(6)a-f display the same preferred conformation. The pyrrolidine and piperidine rings adopt an envelope conformation flattened at N8 and a distorted chair conformation puckered at N8 and flattened at C3, respectively, with the N-substituent in the equatorial position with respect to the piperidine ring. This conformation is similar to that observed for compound 5(6)f in the solid state. From binding studies on the compounds synthesized, compound 5(6)d demonstrated the ability to efficiently displace the binding of [3H]GR65630 to bovine brain area postrema membranes to an extent comparable to MDL 72222. In the von Bezold-Jarisch reflex, compound 5(6)d was equipotent with metoclopramide. It is, therefore, likely that the imidazoline ring may provide a useful bioisosteric replacement for the carbonyl group in 5-HT3 antagonists.


Subject(s)
Imidazoles/chemical synthesis , Serotonin Antagonists/chemical synthesis , Spiro Compounds/chemical synthesis , Animals , Binding, Competitive/drug effects , Cattle , Cerebral Ventricles/metabolism , Crystallization , Female , Imidazoles/pharmacokinetics , Imidazoles/pharmacology , In Vitro Techniques , Indoles/pharmacokinetics , Magnetic Resonance Spectroscopy , Mice , Molecular Conformation , Rats , Reflex/drug effects , Serotonin Antagonists/pharmacokinetics , Serotonin Antagonists/pharmacology , Spiro Compounds/pharmacokinetics , Spiro Compounds/pharmacology , Structure-Activity Relationship , X-Ray Diffraction
15.
J Mol Biol ; 240(3): 267-70, 1994 Jul 15.
Article in English | MEDLINE | ID: mdl-8028010

ABSTRACT

The enzyme encoded by the bgIA gene of Bacillus polymyxa, a type I beta-glucosidase belonging to family I of glycosyl hydrolases, has been purified to homogeneity from an Escherichia coli culture which overexpressed the gene, and crystallized. The crystals, which diffract to 3.0 A resolution, belong to the orthorhombic space group C222(1). The cell dimensions are a = 155.4 A, b = 209.4 A, c = 209.7 A.


Subject(s)
Bacillus/enzymology , beta-Glucosidase/chemistry , Bacillus/genetics , Chromatography, Gel , Chromatography, Ion Exchange , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , beta-Glucosidase/genetics , beta-Glucosidase/isolation & purification
16.
J Pharm Sci ; 82(8): 794-8, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8377116

ABSTRACT

A series of 3 beta-acyloxy-3 alpha-methoxycarbonyltropanes were synthesized and studied by 1H and 13C NMR spectroscopy, and the crystal structure of 3 alpha-methoxycarbonyl-3 beta-pyridincarbonyloxytropane (5d) was determined by X-ray diffraction. In CDCl3 solution, compounds 5a-f display the same preferred conformation. The pyrrolidine and piperidine rings adopt an envelope conformation flattened at N-8 and a distorted chair conformation puckered at N-8 and flattened at C3, respectively, with the N-substituent in the equatorial position with respect to the piperidine ring. The pharmacological profile of one of these compounds makes it an adequate candidate for the design of novel GABAB antagonist agents.


Subject(s)
GABA Antagonists , Tropanes/chemistry , Tropanes/pharmacology , Animals , Crystallography , Guinea Pigs , Magnetic Resonance Spectroscopy , Molecular Conformation , Muscle, Smooth/drug effects , Structure-Activity Relationship , Tropanes/chemical synthesis
17.
Acta Crystallogr A ; 49 ( Pt 2): 306-15, 1993 Mar 01.
Article in English | MEDLINE | ID: mdl-8447986

ABSTRACT

The success of molecular replacement depends, in part, on the degree of similarity of the target and search molecules. We have systematically investigated this effect in cross-rotation functions for members of the aspartic proteinase family of enzymes. The influence of various parameters on peak heights was investigated for six search models using magnitude of F(obs) data for two target enzymes. The beneficial effects of high-resolution data and a large radius of integration are most pronounced when target and search molecules have high-percentage identities. Correction for small differences in domain-domain orientation (typically 4-8 degrees) between search and target structures leads to only a marginal improvement in the rotation-function peak height. There is an almost linear relationship between the structural distance, D, a parameter used in cluster analysis to define differences between three-dimensional protein structures, and the height of the cross-rotation-function peaks.


Subject(s)
Aspartic Acid Endopeptidases/chemistry , Chemical Phenomena , Chemistry, Physical , Computer Simulation , Crystallization , Molecular Structure , X-Ray Diffraction
18.
J Pharm Sci ; 81(1): 94-8, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1619578

ABSTRACT

The vibrational (IR and Raman) and 1H and 13C NMR spectra of the analgesic and anti-inflammatory benzidamine salicylate (Benzasal) have been examined, and the results are described. The crystal structure of this compound has been determined by X-ray diffraction. To assist in interpretation of the spectroscopic data, some measurements of benzidamine, benzidamine hydrochloride, and salicylic acid have also been made. The most important intermolecular interactions of benzidamine salicylate in the solid state are N(+)H...O(-)...-C hydrogen bonds involving both salicylate oxygen atoms (d = 2.658 A and 3.228 A). At least the stronger hydrogen bond remains in CDCl3 solution. Moreover, the strong intramolecular hydrogen bond O-H...O(-)...-C within the salicylate anion has also been observed in the solid state and in solution.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemistry , Benzydamine/chemistry , Anti-Inflammatory Agents , Calorimetry, Differential Scanning , Magnetic Resonance Spectroscopy , Molecular Structure , Spectrophotometry, Infrared , X-Ray Diffraction
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