Enzyme structure and function

We report the cloning and functional charaterization of the full-length cDNA and gene encoding a Toxoplasma gondii DNA repair enzyme designated TgDRE. The gene is composed of three exons separated by two introns of 780 and 630 bp, and encodes a protein with a predicted molecular mass of 49.6 kDa. The native TgDRE protein, with a molecular mass of 60 kDa, is only detected in the virulent tachyzoite stage of T. gondii. However, the transcript is present in both asexual parasite stages, virulent tachyzoite and avirulent encysted bradyzoite. ...

9p research12 01-06-2013 33 5   Download

A severe challenge to the idea that mitochondrial DNA mutations play a major role in the aging process in mammals is that clear loss-of-function mutations accumulate only to very low levels (under 1% of total) in almost any tissue, even by very old age. Their accumulation is punctate: some cells become nearly devoid of wild-type mitochondrial DNA and exhibit no activity for the partly mitochondrially encoded enzyme cytochrome c oxidase. Such cells accumulate in number with aging, suggesting that they survive indefinitely, which is itself paradoxical....

7p research12 01-06-2013 27 3   Download

In addition to kallikrein hK3, a serine protease generally reported as PSA (prostate-speci®c antigen), at least two other enzymes in human seminal plasma also cleave syn-thetic peptidyl substrates derived from the sequence of human semenogelins. We have identi®ed one of these as prostatic acid phosphatase (PAP), a major component of prostatic ¯uid whose physiological function is unclear.

6p research12 29-04-2013 31 4   Download

Recombinant glycerol dehydratase ofKlebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probablya2b2c2 .When(R)- and (S)-propane-1,2-diolswere used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase,an iso-functional enzyme,the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R) /Km(S) ¼1.5). ...

11p research12 23-04-2013 34 2   Download

Short-chaindehydrogenases/reductases (SDRs) are enzymes of great functional diversity. Even at sequence identities of typically only 15–30%, specific sequence motifs are detect-able, reflecting common folding patterns. We have devel-oped a functional assignment scheme based on these motifs and we find five families. Two of these families were known previously and are called
classical and
extended families, but they are now distinguished at a further level based on coenzyme specificities.

9p research12 23-04-2013 38 3   Download

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and Fe II /2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail.

9p research12 23-04-2013 44 4   Download

ThemaltoseATP-binding cassette transporter ofSalmonella typhimurium is composed of a membrane-associated com-plex (MalFGK2) and a periplasmic receptor (MalE). In addition to its role in transport, the complex acts as a repressor ofmaltose-regulatedgene expressionand is subject to inhibition in the process of inducer exclusion. These activities are thought to be mediated by interactions of the ATPase subunit, MalK, with the transcriptional activator, MalT, and nonphosphorylated enzyme IIA of the glucose phosphotransferase system, respectively. ...

12p research12 23-04-2013 35 3   Download

Mammalian adenylate cyclases are predicted to possess complex topologies, comprising two cassettes of six trans-membrane-spanningmotifs followedbyacytosolic, catalytic ATP-binding domain. Recent studies have begun to provide insights on the tertiary assembly of these proteins; crystal-lographic analysis has revealed that the two cytosolic domains dimerize to forma catalytic core, whilemore recent biochemical and cell biological analysis shows that the two transmembrane cassettes also associate to facilitate the functional assembly and trackingof the enzyme. ...

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We have previously reported that two trypsin-like enzymes, acrosin and spermosin, play key roles in sperm penetration through the vitelline coat of the ascidian (Urochordata) Halocynthia roretzi[Sawadaet al. (1984),J. Biol. Chem.259, 2900±2904; Sawadaet al. (1984), Dev. Biol.105, 246±249]. Here, we show the amino-acid sequence of the ascidian preprospermosin, which is deduced from the nucleotide sequence of the isolated cDNA clone.

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The use ofBacillus amyloliquefaciensfor enzyme production and its exceptional highprotein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated. In addition to type I signal peptidases SipS(ba) [Meijer,W.J.J., de Jong,A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J.M. (1995)Mol. Microbiol.17, 621±631] and SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim. Biophys.

12p research12 23-04-2013 34 4   Download

BACE2 (Memapsin 1) is a membrane-bound aspartic pro-tease that is highlyhomologouswithBACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating theb-amyloid peptide and presumably causes Alzheimer’s disease (AD), BACE2 has not been demonstrated to be directly involved in APP pro-cessing, and its physiological functions remain to be deter-mined.In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cyto-solic domains/peptides....

10p tumor12 22-04-2013 25 2   Download

Dnmt3a is ade novoDNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high pre-ference for hemimethylatedDNA. However, Dnmt1 can be activated for themethylationof unmodifiedDNA.We show here that the Dnmt3a andDnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present.

4p tumor12 22-04-2013 34 2   Download

The analysis of the structure and function of long chain-producing polyprenyl diphosphate synthase, which syn-thesizes the side chain of ubiquinone, has largely focused on the prokaryotic enzymes, and little is known about the eukaryotic counterparts. Here we show that decaprenyl diphosphate synthase fromSchizosaccharomyces pombeis comprised of a novel protein named Dlp1 acting in part-nership with Dps1. Dps1 is highly homologous to other prenyl diphosphate synthases but Dlp1 shares only weak homologywithDps1....

9p tumor12 20-04-2013 33 2   Download

CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation ofcinnamicacid to formprecursorsofligninandmanyother phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to iden-tify active site residues likely togovern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directedmutagenesis.

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The gene encodingBacillus subtilisUMP kinase (pyrH/ smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 unitsÆmg )1 of protein. In the absenceofGTP,theactivityofB. subtilisenzyme is less than 10% of its maximum activity. Only dGTP and 3¢-anthraniloyl-2¢-deoxyguanosine-5¢-triphosphate (Ant-dGTP) can increase catalysis significantly.

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Yeast Erv1p is a ubiquitous FAD-dependent sulfhydryl oxidase, located in the intermembrane space of mito-chondria.The dimeric enzyme is essential for survival of the cell.Besides the redox-active CXXC motif close to the FAD, Erv1p harbours two additional cysteine pairs.Site-directed mutagenesis has identified all three cysteine pairs as essential for normal function.The C-terminal cysteine pair is of structural importance as it contributes to the correct arrangement of the FAD-binding fold.

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This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase fromAcinetobacter radioresistensS13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase.

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a-Crystallin, a molecular chaperone and lens structural protein protects soluble enzymes against heat-induced aggregation and inactivation by a variety of molecules. In this study we investigated the chaperone function ofa-crys-tallininamorephysiological systeminwhicha-crystallinwas incorporated into red cell
ghosts. Its ability to protect the intrinsic membrane protein Na/K-ATPase from external stresses was studied. Red cell ghosts were created by lysing the red cells and removing cytoplasmic contents by size-exclusion chromatography. ...

7p fptmusic 16-04-2013 48 2   Download

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofac-tors thiamine diphosphate andmagnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109....

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Aging is accompanied by a gradual deterioration of cell functions. Mitochondrial dysfunction and accumulation of protein damage have been proposed to contribute to this process. The present study was carried out to examine the effects of aging in mitochondrial matrix isolated from rat liver. The activity of Lon protease, an enzyme implicated in the degradation of abnormal matrix proteins, was meas-ured and the accumulation of oxidation and glycoxidation (N e -carboxymethyllysine, CML) products was monitored using immunochemical assays....

8p fptmusic 16-04-2013 26 2   Download