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1.
The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds. 相似文献
2.
Abstract Carbonic anhydrase 2 (CA2) enzyme deficiency caused by CA2 gene mutations is an inherited disorder characterized by symptoms like osteopetrosis, renal tubular acidosis, and cerebral calcification. This study has collected the CA2 deficiency causal missense mutations and assessed their pathogenicity using diverse computational programs. The 3D protein models for all missense mutations were built, and analyzed for structural divergence, protein stability, and molecular dynamics properties. We found M-CAP as the most sensitive prediction method to measure the deleterious potential of CA2 missense mutations. Free energy dynamics of tertiary structure models of CA2 mutants with DUET, mCSM, and SDM based consensus methods predicted only 50% of the variants as destabilizing. Superimposition of native and mutant CA2 models revealed the minor structural fluctuations at the amino acid residue level but not at the whole protein structure level. Near native molecular dynamic simulation analysis indicated that CA2 causative missense variants result in residue level fluctuation pattern in the protein structure. This study expands the understanding of genotype-protein phenotype correlations underlying CA2 variant pathogenicity and presents a potential avenue for modifying the CA2 deficiency by targeting biophysical structural features of CA2 protein. Communicated by Ramaswamy H. Sarma 相似文献
3.
The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds. 相似文献
4.
AbstractNPC1 is a 25-exon gene located on the long arm of chromosome 18q11.2 and encodes NPC1, a transmembrane protein comprising 1278 amino acid residues. Mutations in the NPC1 gene can cause Niemann-Pick disease type C (NP-C), a rare autosomal-recessive neurovisceral disease. We assessed mutant protein folding using computer-based molecular dynamics (MD) simulations and molecular docking of the three most common NPC1 mutations, all of which result in changes in a cysteine-rich luminal loop region of the protein: a) I1061T is the most commonly detected variant in patients with NP-C worldwide; b) P1007A is the second most common variant, frequently detected in Portuguese, British and German patients; c) G992W occurs most often in patients of Acadian descent. Analyses of molecular structural information and related cellular physiological processes revealed that mutant NPC1 proteins exhibited altered function despite being far from the N-terminal domain cholesterol binding. MD simulations revealed that mutant I1061T protein shows remarkable instability in comparison the WT and also de other mutants, and interestingly this mutant has been identified as the most common variant. In the case of the mutant P1007A, it is presumed that this substitution promotes larger structural changes than proline due to their greater hydrophobic properties.Structural changes related to the G992W mutation may affect the physicochemical space of G992W variant protein because tryptophan induces hydrophobic interactions. Cholesterol docking studies focused on binding recognition showed differences in the binding positions of variants versus the wild-type protein that go some way to explaining the molecular pathogenesis.Communicated by Ramaswamy H. Sarma 相似文献
5.
Mutants of Saccharomyces cerevisiae defective in acyl-CoA synthetase (EC 6.2.1.3) were isolated. The mutants were concentrated by the radiation-suicide technique with the use of tritiated palmitic acid. Selection of the mutants was based on the premise that acyl-CoA synthetase activity would become indispensable when yeast cells in which fatty acid synthesis de novo is blocked are grown in a medium supplemented with fatty acid. The mutant strains isolated exhibited low acyl-CoA synthetase activity in vitro. Furthermore, they accumulated markedly more of the incorporated palmitic acid in the nonesterified form than did the wild- type strain. Some of the mutants showed thermosensitive acyl-CoA synthetase activity, indicating a mutation of the structural gene of the enzyme. Genetic studies on these mutants indicated that their phenotype resulted from a single, recessive mutation of a nuclear gene, designated faa 1 (fatty acid activation). 相似文献
6.
AbstractProtection of telomere 1 (POT1) is a key component of shelterin complex, essential for maintaining telomere length and its regulation. It consists of N-terminal domain (residues 1–299), which interacts with telomeric ssDNA, and the C-terminal domain (residues 320–634) that binds to the tripeptidyl-peptidase I (TPP1). A large number of naturally occurring mutations in the POT1 gene are associated with glioma, cardiac angiosarcoma and cutaneous familial melanoma (FM). In particular, Q94E mutation disrupts the interaction of POT1 with telomeric DNA which subsequently enhances telomere uncapping and elongation and promotes the development of cutaneous familial melanoma. To understand the underlying mechanism of familial melanoma developed by Q94E-mutation, we have performed extensive structure analysis of WT and mutant protein followed by molecular dynamics simulations. Q94E mutation causes a dramatic change in the structure and stability of POT1 protein. A considerable decrease in the flexibility, fluctuation and solvent accessibility of Q94E was observed in comparison to the WT, indicating overall destabilization of protein. Essential dynamics and Anisotropic Network Mode analysis have quantified a significant change in direction and magnitude of conformational motion in Q94E mutant compared to WT. A significant loss of frustration due to Q94E mutation was also observed. Our findings indicate the loss of protein stability and dynamics of POT1 protein by Q94E mutation may be associated with the familial melanoma. Abbreviations ANM anisotropic network mode ED essential dynamics FM familial melanoma MD molecular dynamics POT1 protection of telomere 1 Rg radius of gyration RMSD root-mean-square deviation RMSF root-mean-square fluctuations SASA solvent accessible surface area SIFT sorting Intolerant from Tolerant TPP1 tripeptidyl-peptidase I WT wild type Communicated by Ramaswamy H. Sarma 相似文献
7.
Summary A temperature-sensitive mutant of Escherichia coli was identified as having an altered alanyl-tRNA synthetase. Specific activity of wild type and mutant cell-free extracts showed no difference in the hydroxamate assay; the charging activity, however, was more than 10 fold lower for mutant extract protein. Wild type alanyl-tRNA synthetase has been purified 344 fold, the mutant enzyme was enriched 45 fold. With these preparations the following results were obtained:Sedimentation analysis in sucrose gradients indicates a molecular weight of the mutant enzyme of half the size of the wild type enzyme. Analytical gel filtration yields an approximate size for the native enzyme of 165000 and for the mutant enzyme material of 95,000. The mutant alanyl-tRNA synthetase differs from the wild type enzyme by a 10 fold increase in the k
mfor tRNA; no true difference in the k
m-values for the other substrates was detected. Temperature studies indicate an unusual low temperature-optimum for the charging reaction of both enzymes, whereas hydroxamate fromation capacity increases linearly up to almost 50°C. High temperature treatment of the native enzyme selectively affects the aminoacylation reaction but not the activation step; no effect of such treatment of the mutant enzyme was detected. It is proposed that the mutation causes the enzyme to dissociate and that the resulting subunits possess and altered tRNA binding site. 相似文献
9.
PurposeHyperglycemia causes abnormal accumulation of methylglyoxal (MGO) and concomitant DNA, protein glycation. These pathophysiological changes further leads to diabetic complications. Yeast Saccharomyces cerevisiae is one of the best model to study MGO-induced glycation modifications. The aim of the present study was to investigate the effect of MGO on protein, DNA glycation, and oxidative stress markers using S. cerevisiae as a system. MethodsSaccharomyces cerevisiae cells were incubated with 8 mM of MGO for 4 h and 24 h. After incubation, protein and DNA samples were isolated from the lysed cells. The samples were analyzed for various glycation (fructosamine, β-amyloid, free amino group, free thiol group, and hyperchromic shift analysis) and oxidative stress markers (total antioxidant potential, catalase, glutathione, and lipid peroxidation). ResultsMGO (8 mM) acted as a potent glycating agent, causing protein and DNA glycation in treated yeast cells. The glycation markers fructosamine and β-amyloid were significantly elevated when incubated for 4 h as compared to 24 h. Oxidative stress in the glycated yeast cells alleviated cellular antioxidant capacity and reduced the cell viability. ConclusionMGO caused significant glycation modifications of proteins and DNA in yeast cells. It also triggered increase in intracellular oxidative stress. MGO-induced protein, DNA glycation, and oxidative stress in S. cerevisiae indicate the suitability of the yeast model to study various biochemical pathways involved in diabetic complications and even conformational pathologies. 相似文献
10.
AbstractThe fungal chitinase I obtained from Thermomyces lanuginosus SSBP, a thermophilic deuteromycete, has an optimum growth temperature and pH of 323.15 K and 6.5, respectively . This enzyme plays an important task in the defence mechanism of organisms against chitin-containing parasites by hydrolysing β-1, 4-linkages in chitin. It acts as both anti-fungal and biofouling agents, with some being thermostable and suitable for the industrial applications. Three-dimensional model of chitinase I enzyme was predicted and analysed using various bioinformatics tools. The structure of chitinase I exhibited a well-defined TIM barrel topology with an eight-stranded α/β domain. Structural analysis and folding studies at temperatures ranging from 300 to 375 K using 10 ns molecular dynamics simulations clearly showed the stability of the protein was evenly distributed even at higher temperatures, in accordance with the experimental results. We also carried out a number of 20 ns constant pH molecular dynamics simulations of chitinase I at a pH range 2–6 in a solvent. This work was aimed at establishing the optimum activity and stability profiles of chitinase I. We observed a strong conformational pH dependence of chitinase I and the enzyme retained their characteristic TIM barrel topology at low pH. 相似文献
11.
An extracellular endo-polygalacturonase (PGase) produced by a mutant of Saccharomyces cerevisiae was isolated. The enzyme was regarded, immunologically, as a PGase belonging to the Kluyveromyces marxianus group. The enzyme had properties similar to the PGase from K. marxianus in heat and pH stability, and N-terminal amino acid sequence. However, the enzyme showed different properties in optimum pH and temperature, molecular weight, and reactivity in antiserum against PGase from K. marxianus, indicating that the enzyme has a different molecular structure from the PGase from K. marxianus. 相似文献
12.
AbstractDuring directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs 610T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs 610T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs 610T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates. 相似文献
13.
Abstract Cytochrome P450cam is capable of reductively dehalogenating several chlorinated alkanes at low, but measurable, rates. In previous investigations of structure-function relationships in this enzyme using molecular dynamics simulations, we noticed that 1,1,1-trichloroethane (TCA) exhibits a very high degree of mobility in the active site due to its smaller molecular volume relative to the native substrate, camphor(1,2). Several amino acid sidechains lining the active site also exhibit significant dynamic fluctuations, possibly as a result of poor steric complementarity to TCA. Guided by these results, we modeled double (F87W, T185F) and triple (F87W, T185F, V295I) mutants of P450cam, which provide additional bulk in the active site and increase the frequency of heme-substrate collision. Molecular dynamics simulations (300 ps on each protein) indicate that these mutants do not significantly perturb the three-dimensional fold of the enzyme, or local structure in the region of the active site. Both mutants bind the substrate more stably near the heme than the wild-type. Interestingly, however, the bulkier triple mutant seems to actually inhibit heme-substrate interactions relative to the double mutant. Over the final 200 ps of simulation, TCA is within 1 Å of nonbonded contact with the heme 25% more often in the double mutant versus the wild-type. The triple mutant, on the other hand, binds TCA within 1 Å of the heme only 15% as often as the wild-type. These results indicate that the double mutant may reductively dehalogenate TCA, a property not observed for the native protein. Implications for other experimentally measurable parameters are discussed. 相似文献
14.
ABSTRACTThe autophagy receptor for selective reticulophagy, RETREG1/FAM134B is essential for ER maintenance, and its dysfunction is associated with neuronal disorders, vascular dementia, or viral infections. The protein consists of the reticulon-homology domain (RHD) that is flanked at the N- and C-termini by an intrinsically disordered protein region (IDPR), where the C terminal IDPR carries the indispensable LC3-interacting region (LIR) motif for the interaction with LC3. The RHD of RETREG1 is presumed to play a role in membrane remodeling, but the absence of a known 3D structure of this domain so far prevented researchers from gaining mechanistic insights into how the RETREG1 RHD curves membranes, and thereby facilities reticulophagy. The recent study by Bhaskara et al., which is described in this editor’s corner article, used molecular dynamics (MD) simulations to create a structural model of the RETREG1 RHD. MD simulations along with in vitro liposome remodeling experiments reveal how the RHD domain acts on the ER membrane and, in concert with the C terminal IDPR, executes the function of RETREG1 in selective reticulophagy. Abbreviations: ER, endoplasmic reticulum; IDPR, intrinsically disordered protein region; LIR, LC3-interacting region; MD, molecular dynamics; RHD, reticulon-homology domain; TM, transmembrane 相似文献
15.
Abstract In the absence of an experimentally elucidated three-dimensional structure of the human CDA, we built an homology model of this enzyme starting from the crystal structure of its E. coli homologous. Furthermore, we docked in the active site alternatively the substrate, the intermediate or the product. By means of molecular dynamics simulations, we determined the topology of the active site, identifying the amino acids involved in the catalytic mechanism, and outlining the central role played by E67. 相似文献
16.
PurposeA mutant BA-SacB-Del encoding BA-SacB minus K296-D320 region was constructed to analyze its effect on catalytic characteristics of the enzyme as well as help deepen understanding of the catalytic mechanism of BA-SacB and even proteins of GH68 family. MethodsBased on the comparison of levansucrases from Bacillus amyloliquefaciens (BA-SacB) and Sphingopyxis macrogoltabida (SM-Lev), a mutant BA-SacB-Del encoding BA-SacB minus K296-D320 region was constructed and its effect on catalytic characteristics of the enzyme was analyzed. ResultsDeletion of this region would undoubtedly affect the conserved structure (i.e., central negatively charged cavity surrounded by five antiparallel β-strands) shared by the GH68 family. Therefore, Escherichia coli-expressing mutant BA-SacB-Del could more efficiently catalyze the production of levan in media containing high concentration of sucrose, which is unrealizable for BA-SacB. ConclusionsThis result should be valuable for understanding this conditional lethal mechanism. Therefore, this study should be very valuable for understanding the catalytic mechanism of BA-SacB and even proteins of the GH68 family. More importantly, levan can be conveniently produced by direct fermentation of sucrose-containing media with E. coli-expressing BA-SacB-Del which is not sensitive to sucrose. 相似文献
17.
AbstractInvertases are used for several purposes; one among these is the production of fructooligosaccharides. The aim of this study was to biochemically characterize invertase from industrial Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa isolated from Cerrado soil. The optimum pH and temperature were 4.0 and 70?°C for Rhodotorula mucilaginosa invertase and 4.5 and 50?°C for Saccharomyces cerevisiae invertase. The pH and thermal stability from 3.0 to 10.5 and 75?°C for R. mucilaginosa invertase, respectively. The pH and thermal stability for S. cerevisiae CAT-1 invertase from 3.0 to 7.0, and 50?°C, respectively. Both enzymes showed good catalytic activity with 10% of ethanol in reaction mixture. The hydrolysis by invertases occurs predominantly when sucrose concentrations are ≤5%. On the other hand, the increase in the concentration of sucrose to levels above 10% results in the highest transferase activity, reaching about 13.3?g/L of nystose by S. cerevisiae invertase and 12.6?g/L by R. mucilaginosa invertase. The results demonstrate the high structural stability of the enzyme produced by R. mucilaginosa, which is an extremely interesting feature that would enable the application of this enzyme in industrial processes. 相似文献
18.
AbstractHuman meprin-β, a zinc metalloprotease belonging to the astacin family, have been found to be associated with many pathological conditions like inflammatory bowel disease, fibrosis and neurodegenerative disease. The inhibition of meprin-β by various inhibitors, both macromolecular and small molecules, is crucial in the control of several diseases. Human fetuin-A, a negative acute phase protein involved in inflammatory disease, has recently been identified as an endogenous inhibitor for meprin-β. In this computational study, an integrated in silico approach was performed using existing structural information of meprin-β coupled with ab initio modelling of human fetuin-A to predict a rational model of the complex through protein–protein docking. Further, the models were optimized and validated to generate an ensemble of conformations through extensive molecular dynamics simulation. Virtual alanine scanning mutagenesis was explored to identify hotspot residues on both proteins significant for protein–protein interaction (PPI). The results of the study provide structural insight into PPI between meprin-β and fetuin-A which can be useful in designing molecules to modulate meprin-β activity. Communicated by Ramaswamy H. Sarma 相似文献
19.
Abstract The crystal structure of Rv0098, a long-chain fatty acyl-CoA thioesterase from Mycobacterium tuberculosis with bound dodecanoic acid at the active site provided insights into the mode of substrate binding but did not reveal the structural basis of substrate specificities of varying chain length. Molecular dynamics studies demonstrated that certain residues of the substrate binding tunnel are flexible and thus modulate the length of the tunnel. The flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. A combination of crystallographic and molecular dynamics studies thus explained the structural basis of accommodating long chain substrates by Rv0098 of M. tuberculosis. 相似文献
20.
Abstract Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125000, and two molecules of its cognate tRNA (M r = 24160) cocrystallize in the cubic space group 1432 (a = 354 Å). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D 2O content in order to use the contrast variation method to distinguish between the protein and tRNA The synthetase was first located at 40 Å resolution using the 65% D 2O neutron data (tRNA matched). tRNA molecules were found at 20 Å resolution using both neutron and X-ray data. The resulting model was refined against 10 Å resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure, solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents. 相似文献
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