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Classically, alpha-1,4-glucan synthases have been divided into two families, animal/fungal glycogen synthases (GS) and bacterial/plant starch synthases (G(S)S), according to differences in sequence, sugar donor specificity and regulatory mechanisms. Detailed sequence analysis, predicted secondary structure comparison and threading analysis show that these two families are structurally related and that some domains of GSs were acquired to meet regulatory requirements. Archaeal G(S)S present structural and functional features that are conserved in one, the other or both families. Therefore, they are the link between GS and G(S)S and harbor the minimal sequence and structural features that constitute the minimum catalytic unit of the alpha-1,4-glucan synthase superfamily.  相似文献   

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Recent data suggest the source of F(0)F(1) ATP synthase determines a significant and surprising difference in the size of a putative rotating ring of integral membrane subunits of F(0); this can be correlated with biochemical data suggesting there is variation in the number of protons translocated per ATP synthesised.  相似文献   

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Glycogen synthase kinase 3β (GSK-3β) is a serine-threonine kinase belonging to the CMGC family that plays a key role in many biological processes, such as glucose metabolism, cell cycle regulation, and proliferation. Like most protein kinases, GSK-3β is regulated via multiple pathways and sites. We performed all-atom molecular dynamics simulations on the unphosphorylated and phosphorylated unbound GSK-3β and the phosphorylated GSK-3β bound to a peptide substrate, its product, and a derived inhibitor. We found that GSK-3β autophosphorylation at residue Tyr(216) results in widening of the catalytic groove, thereby facilitating substrate access. In addition, we studied the interactions of the phosphorylated GSK-3β with a substrate and peptide inhibitor located at the active site and observed higher affinity of the inhibitor to the kinase. Furthermore, we detected a potential remote binding site which was previously identified in other kinases. In agreement with experiments we observed that binding of specific peptides at this remote site leads to stabilization of the activation loop located in the active site. We speculate that this stabilization could enhance the catalytic activity of the kinase. We point to this remote site as being structurally conserved and suggest that the allosteric phenomenon observed here may occur in the protein kinase superfamily.  相似文献   

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Non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of the cyclo-oxygenase (COX)-1 and -2 activities of prostaglandin G/H synthase-1 and -2, respectively. They have been extensively used in the treatment of prostaglandin E(2)-mediated chronic inflammatory diseases. Selective COX-2 inhibitors (coxibs), which were developed to provide an alternative with reduced gastrointestinal risk for the traditional NSAIDs, have been associated with an increased incidence of major adverse cardiovascular events. Could the targeting of microsomal prostaglandin E(2) synthase (mPGES-1) lead to novel anti-inflammatory drugs with possibly reduced risks of gastrointestinal and cardiovascular side effects?  相似文献   

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The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate l-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase complex, together with the observed low affinity of sTrpB2i for l-serine, couples the rate of tryptophan biosynthesis in S. solfataricus to the cytosolic availability of l-serine.  相似文献   

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Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S](2+) clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either [4Fe-4S](2+), [4Fe-4S](+), or a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe M?ssbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. A mixed [4Fe-4S](2+):[2Fe-2S](2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.  相似文献   

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The rapid and specific determination of picomole quantities of δ-aminolevulinate has been accomplished by its specific enzymatic conversion to uroporphyrinogen I and fluorometric detection of the oxidized uroporphyrin I. The coupled enzyme assay was linear with time and protein concentration and required less than 3 h for 25 individual determinations. Under the standard assay conditions, 1 to 100 pmol of uroporphyrin I was reliably quantitated; these values corresponded to a range of ALA synthase activities from 0.15 to 15 nmol/h/ml of enzyme. The sensitivity of this method was comparable to the more time-consuming radiochemical determinations of ALA synthase. In addition, this method was at least 10 times more sensitive than the colorimetric assays for ALA synthase activity. The rapidity, specificity, and sensitivity of this new method make it useful for monitoring the purification of ALA synthase and for reliable determinations of low levels of ALA synthase activity in crude tissue or cultured cell homogenates.  相似文献   

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A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.  相似文献   

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Nurhayati N  Ober D 《Phytochemistry》2005,66(11):1346-1357
Quinolizidine alkaloids are the most prominent group of alkaloids occurring in legumes, except for many members of the tribe Crotalarieae that accumulate pyrrolizidine alkaloids (PAs). To study the evolution of PA biosynthesis as a typical pathway of plant secondary metabolism in this tribe, we have searched for a cDNA coding for homospermidine synthase (HSS), the enzyme catalyzing the first specific step in this biosynthesis. HSS was shown to have been recruited from deoxyhypusine synthase (DHS) by independent gene duplication in several different angiosperm lineages during evolution. Except for a cDNA sequence coding for the DHS of Crotalaria retusa, no data is available concerning the origin of PA biosynthesis within this tribe of the Fabaceae. In addition to several pseudogenes, we have identified one functional DHS in C. scassellatii and two in C. juncea. Despite C. juncea plants under study being devoid of PAs, we have found that the two sequences of C. juncea are different with respect to their genomic organization, their tissue-specific expression, and their biochemical activities. Supported by the branching pattern of a maximum likelihood analysis of these sequences, they have been classified as "class 1" and "class 2" DHS. It remains open whether the duplicated DHS belonging to class 2 is involved in the biosynthesis of PAs.  相似文献   

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Morikawa T  Yasuno R  Wada H 《FEBS letters》2001,498(1):16-21
Lipoic acid is a coenzyme essential to the activity of enzymes such as pyruvate dehydrogenase, which play important roles in central metabolism. However, neither the enzymes responsible for biosynthesis nor the biosynthetic event of lipoic acid has been reported in mammalian cells. In this study, a mouse mLIP1 cDNA for lipoic acid synthase has been identified. We have shown that the cDNA encodes a lipoic acid synthase by its ability to complement a mutant of Escherichia coli defective in lipoic acid synthase and that mLIP1 is targeted into the mitochondria. These findings suggest that mammalian cells are able to synthesize lipoic acid in mitochondria.  相似文献   

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The enzyme lumazine synthase (LS) catalyzes the penultimate step of riboflavin biosynthesis in plants, fungi and bacteria. The quaternary structure of the polypeptide differs between species, existing as pentamers or as icosahedrally arranged dodecamers of pentamers with 60 subunits. The pathogen Brucella spp. expresses two proteins that exhibit LS activity, RibH1 and RibH2. The latter enzyme belongs to a novel third category of quaternary arrangement for LS, that of a decameric structure assembled as a head-to-head oriented dimer of pentamers. In contrast, the RibH1 enzyme is assembled as a pentamer, as noted for several other LS enzymes. RibH1 appears to be the functional LS in Brucella spp., whereas RibH2, an enzyme of lower catalytic activity, is a virulence factor presumably acting in response to oxidative stress. The latter observation prompted us to further investigate the structural and catalytic properties of RibH2 in order to clarify the biological function of this enzyme. Here, we present a detailed analysis of two new crystallographic forms of RibH2 that explain the low catalytic activity of this enzyme in comparison with RibH1 and other LSs. Additionally, we analyze the effect of pH on the structure of this enzyme, and the binding of riboflavin and 6,7-dimethyl-8-ribityllumazine to its active site.  相似文献   

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beta-Cyanoalanine synthase (CAS; EC 4.4.1.9) and two kinds of cysteine synthases (CS; EC 4.2.99.8) have been purified from the particulate fraction of potato tubers. By DEAE Sephacel and Resource PHE chromatography, CAS activity was separated from two CS activities, designated as CS-1 and CS-2. The molecular masses of CAS, CS-1 and CS-2 were estimated to be 37, 39 and 34 kDa, respectively, by SDS-PAGE analysis. The purified CAS had CS activity, and both CS-1 and CS-2 had CAS activity. However, CAS and CSs had significant differences in kinetic characters. The antibody raised against purified CAS discriminated CAS from CSs, whereas the antibody raised against purified CS-2 recognized CS-1 and CS-2 but not CAS. The molecular mass and the partial amino acid sequence of CS-2 were similar to those of the cytosolic CS of potato, whereas the molecular mass of CS-1 was similar to that of the plastidic CS. The partial amino acid sequence of CAS was similar to those of CS isozymes, especially the mitochondrial CS isolated from spinach.  相似文献   

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