Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The crystal structure of thioredoxin from Escherichia coli has been refined by the stereochemically restrained least-squares procedure to a crystallographic R-factor of 0.165 at 1.68 A resolution. In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A and for angle distances 0.035 A. The structure contains 1644 protein atoms from two independent molecules, two Cu2+, 140 water molecules and seven methylpentanediol molecules. Ten residues have been modeled in two alternative conformations. E. coli thioredoxin is a compact molecule with 90% of its residues in helices, beta-strands or reverse turns. The molecule consists of two conformational domains, beta alpha beta alpha beta and beta beta alpha, connected by a single-turn alpha-helix and a 3(10) helix. The beta-sheet forms the core of the molecule packed on either side by clusters of hydrophobic residues. Helices form the external surface. The active site disulfide bridge between Cys32 and Cys35 is located at the amino terminus of the second alpha-helix. The positive electrostatic field due to the helical dipole is probably important for stabilizing the anionic intermediate during the disulfide reductase function of the protein. The more reactive cysteine, Cys32, has its sulfur atom exposed to solvent and also involved in a hydrogen bond with a backbone amide group. Residues 29 to 37, which include the active site cysteine residues, form a protrusion on the surface of the protein and make relatively fewer interactions with the rest of the structure. The disulfide bridge exhibits a right-handed conformation with a torsion angle of 81 degrees and 72 degrees about the S-S bond in the two molecules. Twenty-five pairs of water molecules obey the noncrystallographic symmetry. Most of them are involved in establishing intramolecular hydrogen-bonding interactions between protein atoms and thus serve as integral parts of the folded protein structure. Methylpentanediol molecules often pack against the loops and stabilize their structure. Cu2+ used for crystallization exhibit a distorted octahedral square bipyramid co-ordination and provide essential packing interactions in the crystal. The two independent protein molecules are very similar in conformation but distinctly different in atomic detail (root-mean-square = 0.94 A). The differences, which may be related to the crystal contacts, are localized mostly to regions far from the active site.
J Mol Biol 1990 Mar 05
PMID:Crystal structure of thioredoxin from Escherichia coli at 1.68 A resolution. 218 Nov 45

Studies with biomimetic models can yield considerable insight into mechanisms of enzymatic catalysis. The discussion above indicates how such information has been important in the cases of flavoproteins, hemoproteins, and, to a lesser extent, the copper protein dopamine beta-hydroxylase. Some of the moieties that we generally accept as intermediates (i.e., high-valent iron oxygen complex in cytochrome P-450 reactions) would be extremely hard to characterize were it not for biomimetic models and more stable analogs such as peroxidase Compound I complexes. Although biomimetic models can be useful, we do need to keep them in perspective. It is possible to alter ligands and aspects of the environment in a way that may not reflect the active site of the protein. Eventually, the model work needs to be carried back to the proteins. We have seen that diagnostic substrates can be of considerable use in understanding enzymes and examples of elucidation of mechanisms through the use of rearrangements, mechanism-based inactivation, isotope labeling, kinetic isotope effects, and free energy relationships have been given. The point should be made that a myriad of approaches need to be applied to the study of each enzyme, for there is potential for misleading information if total reliance is placed on a single approach. The point also needs to be made that in the future we need information concerning the structures of the active sites of enzymes in order to fully understand them. Of the enzymes considered here, only a bacterial form of cytochrome P-450 (P-450cam) has been crystallized. The challenge to determine the three-dimensional structures of these enzymes, particularly the intrinsic membrane proteins, is formidable, yet our further understanding of the mechanisms of enzyme catalysis will remain elusive as long as we have to speak of putative specific residues, domains, and distances in anecdotal terms. The point should be made that there is actually some commonality among many of the catalytic mechanisms of oxidation, even among proteins with different structures and prosthetic groups. Thus, we see that cytochrome P-450 has some elements of a peroxidase and vice versa; indeed, the chemistry at the prosthetic group is probably very similar and the overall chemistry seems to be induced by the protein structure. The copper protein dopamine beta-hydroxylase appears to proceed with chemistry similar to that of the hemoprotein cytochrome P-450 and, although not so thoroughly studied, the non-heme iron protein P. oleovarans omega-hydroxylase.(ABSTRACT TRUNCATED AT 400 WORDS)
Crit Rev Biochem Mol Biol 1990
PMID:Enzymatic oxidation of xenobiotic chemicals. 218 70

Thymulin (formerly called FTS) is a well-defined nonapeptide hormone produced by thymic epithelial cells. Its biological activity and antigenicity depend on the presence of the metal zinc in the molecule. The interaction between this metal ion and thymulin has been investigated by means of one- and two-dimensional NMR experiments. These experiments were performed in dimethyl-d6 sulfoxide solution and in aqueous medium with different metal:peptide ratios. The results are compared with those obtained for complexes of thymulin with other metal ions (Cu2+ and Al3+) and for the [Ala4]- and [Ala8]-analogs in terms of biological activity. These comparative studies suggest that the 1:1 complex is the only conformation recognized by the antibodies. From the NOESY data, a spatial conformation has been proposed for this complex. This conformation should be the physiological one and could lead to a better insight into the conformation requirements at receptor sites.
Mol Chem Neuropathol 1990 Jan
PMID:Structural and conformational analysis of metal-containing peptides by one- and two-dimensional NMR spectroscopy. The case of thymulin. 227 5

In designing new DNA recognizing and cleaving reagents, we introduce herein a bisacridine derivative (referred to as bisacridine) in which two acridine heterocycles are connected by a penta(ethylene glycol) bridging chain. This compound offers two possible functions: 1, stabilization of DNA bisacridine intercalator complex by metal ion. The penta(ethylene glycol) chain stabilizes metal ions binding to the phosphate site of DNA, where the penta(ethylene glycol) chain constitutes a part of a pseudomacrocyclic ligand for metal binding; and 2, enhancement of metal-assisted hydrolytic cleavage of DNA by means of a metal concentration effect by the pseudomacrocyclic ethereal chain. The binding isotherms of bisacridine with DNA in the presence of metal ions showed that the binding was mainly governed by the cation exchange reaction on the anionic DNA polymer chain, i.e., the exchange between metal ions and the cationic bisacridine. The bisacridine showed an increase DNA binding ability compared to quinacrine, the monoacridine counterpart, and caused an enhancement of DNA cleavage in the presence of Cu2+ ions. Additional experiments which included DNase 1 footprinting in the presence of bisacridine and the DNA cleavage by Cu2+/bisacridine using a 32P end-labelled DNA fragment, suggested that the Cu2(+)-assisted DNA cleavage sites in the presence of bisacridine were in reasonable overlap with the DNA binding sites of bisacridine.
J Mol Recognit 1990 Aug
PMID:Cleavage of double helical DNA by Cu2+ ion in the presence of bisintercalator containing penta(ethylene glycol) connector chain. 227 32

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the production of alpha-amidated peptides from their glycine-extended precursors, a posttranslational modification often required for full biological activity. We have previously cloned cDNAs encoding a 108-kDa bovine PAM precursor. To confirm that this cDNA encodes a functional alpha-amidating enzyme and to begin to examine the structural requirements for the biosynthesis of an active PAM enzyme, we constructed expression vectors that placed the cDNA for either the full-sized enzyme or a form truncated at the carboxyl-terminal (and thus lacking the transmembrane domain) under the control of the mouse metallothionein-1 promoter. We used the resultant plasmids to transfect AtT-20 mouse anterior pituitary corticotrope cells and selected stable lines that expressed increased levels of PAM activity. Transfected cells in which expression from the metallothionein promoter had been induced had up to 15-fold higher levels of PAM mRNA and up to 7.5-fold higher levels of PAM activity than wild-type cells. The PAM activity in the transfected cells shared many enzymatic characteristics with PAM-B, a 38-kDa soluble form of PAM purified from bovine neurointermediate pituitary. These included copper- and ascorbate-dependent activity, an alkaline pH optimum for the peptide substrate D-Tyr-Val-Gly, similar affinities for several other synthetic substrates, and comparable apparent size during gel filtration. Compared to extracts of wild-type cells, extracts from transfected cells showed increased production of five different amino acid alpha-amides. These data indicate that a single enzyme can act on a variety of peptide substrates, and that the full structure of the PAM precursor is not necessary during biosynthesis for expression of active PAM enzyme.
Mol Endocrinol 1990 Jan
PMID:Stable expression of full-length and truncated bovine peptidylglycine alpha-amidating monooxygenase complementary DNAs in cultured cells. 232 63

Immunogenicity of soluble protein antigens in the complexes with synthetic polyions may be regarded as depending both on the nature of polymer carrier and the structure of the protein-polyelectrolyte complex. The immunogenicity of stable soluble complexes of ovalbumin (OA) with polycation - quaternized poly-4-vinylpyridine (C-1) and copolymer of acrylic acid and 2-methyl-5-vinylpyridine (C-2) have been evaluated. Immunization of mice by C-1 have induced a vigorous formation of the anti-OA IgG antibodies and IgE homocytotropic antibodies, while immunogenicity of OA in C-2 was comparable with that of OA alone. The analysis of the structural-chemical features of the complexes investigated has shown that enhanced immunogenicity of C-1 may be due to (1) the non-homogeneous distribution of protein globulae among polycation macromolecules and to (2) the formation of complex with an asymmetrical structure, to (3) the high ability of C-1 to adsorb on a surface of the lymphoid cells and to induce a formation of intercellular aggregates. An enhancing of a stability and a size of C-2 in the presence of Cu2+ shows no influence on a immunogenicity of OA. An immunogenicity of both types of complexes does not depend upon the access of determinants of OA to antibodies so far as it has been shown that complex formation in both cases are not accompanied by an alteration of antigenicity and allergenicity of OA.
Mol Biol (Mosk)
PMID:[The effect of structural-chemical characteristics of water-soluble polyelectrolyte complexes of ovalbumin on their immunological properties]. 236 87

The ACE1 protein of Saccharomyces cerevisiae was expressed as a trpE-ACE1 fusion protein in Escherichia coli and shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. These binding sites contain within them the sequence 5'-TC(T)4-6GCTG-3', which we propose constitutes an important part of the ACE1 consensus recognition sequence.
Mol Cell Biol 1990 Jan
PMID:ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences. 240 47

On the basis of the spatial structure of ascorbate oxidase [Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & Finazzi-Agro, A. (1989) J. Mol. Biol. 206, 513-529], an alignment of the amino acid sequence of the related blue oxidases, laccase and ceruloplasmin is proposed. This strongly suggests a three-domain structure for laccase closely related to ascorbate oxidase and a six-domain structure of ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationships suggest that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and 3 and ceruloplasmin has mononuclear copper ions in domains 2, 4 and 6 and a trinuclear copper between domains 1 and 6.
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PMID:The blue oxidases, ascorbate oxidase, laccase and ceruloplasmin. Modelling and structural relationships. 240 64

The efficiency of Escherichia coli nucleic acids samples: covalently closed circular DNA, linear chromosomal DNA, total RNA degradation mediated by the action of high oxygen pressure; hydrochloric hydroxylamine in alkaline conditions in the presence of cooper ions and in analogous conditions without cooper ions was studied. The nativity of nucleic acids was determined by means of fluorometric analysis of nucleic acids/ethidium bromide complexes. Experiments revealed, that the destructive effect of active oxygen species decreased in the following order: NH2OH.HCl in alkaline conditions in the presence of copper ions-NH2.HCl in alkaline conditions--high pressure of pure oxygen. The stability of nucleic acids decreased in the following order: covalently closed circular DNA-linear DNA-RNA.
Mol Biol (Mosk)
PMID:[Degradation of nucleic acids during generation of superoxide-anion in the presence of copper ions]. 245 81

The pathogenic neisseriae, Neisseria gonorrhoeae and Neisseria meningitidis, possess an outer membrane protein, H.8, which contains a conserved monoclonal antibody (MAb)-binding epitope in all strains tested. We have cloned and sequenced a meningococcal H.8 gene, and determined the characteristics of the predicted protein. The predicted signal peptide has features characteristic of a prokaryotic lipoprotein. The region at the N-terminal end of the mature protein (39 amino acids) is primarily composed of alanine, glutamate and proline residues arranged in imperfect repeats with the consensus sequence AAEAP. The epitope for H.8 MAb-binding was localized to a 20-amino-acid sequence within this region. The remainder of the predicted amino acid sequence shows extensive homology to azurins, which are small blue copper-binding proteins found in a limited number of species of pathogenic bacteria.
Mol Microbiol 1987 Sep
PMID:Localization of a conserved epitope and an azurin-like domain in the H.8 protein of pathogenic Neisseria. 245 58


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