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131.
Role of active‐site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C‐S lyase
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Alessandra Astegno Alessandra Allegrini Stefano Piccoli Alejandro Giorgetti Paola Dominici 《Proteins》2015,83(1):78-90
In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C‐S lyase from Corynebacterium diphtheriae is a pyridoxal 5′‐phosphate‐dependent enzyme in the transsulfuration pathway that catalyzes the α,β‐elimination of sulfur‐containing amino acids, such as l ‐cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L ‐methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active‐site residues, Y55F and Y114F, were characterized by UV‐visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady‐state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130‐fold decrease in at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP‐binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of l ‐cystathionine, and causes a change in substrate specificity towards l ‐serine and O‐acetyl‐l ‐serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C‐S lyase, which seems to be regulated by active‐site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors. Proteins 2015; 83:78–90. © 2014 Wiley Periodicals, Inc. 相似文献
132.
Christine Gersch Sergiu P. Palii Witcha Imaram Kyung Mee Kim S. Ananth Karumanchi Alexander Angerhofer 《Nucleosides, nucleotides & nucleic acids》2013,32(2):118-149
Hyperuricemia is associated with hypertension, metabolic syndrome, preeclampsia, cardio-vascular disease and renal disease, all conditions associated with oxidative stress. We hypothesized that uric acid, a known antioxidant, might become prooxidative following its reaction with oxidants; and, thereby contribute to the pathogenesis of these diseases. Uric acid and 1,3-15N2-uric acid were reacted with peroxynitrite in different buffers and in the presence of alcohols, antioxidants and in human plasma. The reaction products were identified using liquid chromatography-mass spectrometry (LC-MS) analyses. The reactions generate reactive intermediates that yielded triuret as their final product. We also found that the antioxidant, ascorbate, could partially prevent this reaction. Whereas triuret was preferentially generated by the reactions in aqueous buffers, when uric acid or 1,3-15N2-uric acid was reacted with peroxynitrite in the presence of alcohols, it yielded alkylated alcohols as the final product. By extension, this reaction can alkylate other biomolecules containing OH groups and others containing labile hydrogens. Triuret was also found to be elevated in the urine of subjects with preeclampsia, a pregnancy-specific hypertensive syndrome that is associated with oxidative stress, whereas very little triuret is produced in normal healthy volunteers. We conclude that under conditions of oxidative stress, uric acid can form reactive intermediates, including potential alkylating species, by reacting with peroxynitrite. These reactive intermediates could possibly explain how uric acid contributes to the pathogenesis of diseases such as the metabolic syndrome and hypertension. 相似文献
133.
Mammalian spermatogenesis is a complex developmental program in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. One intriguing aspect of sperm production is the dynamic change in membrane lipid composition that occurs throughout spermatogenesis. Cholesterol content, as well as its intermediates, differs vastly between the male reproductive system and nongonadal tissues. Accumulation of cholesterol precursors such as testis meiosis-activating sterol and desmosterol is observed in testes and spermatozoa from several mammalian species. Moreover, cholesterogenic genes, especially meiosis-activating sterol-producing enzyme cytochrome P450 lanosterol 14α-demethylase, display stage-specific expression patterns during spermatogenesis. Discrepancies in gene expression patterns suggest a complex temporal and cell-type specific regulation of sterol compounds during spermatogenesis, which also involves dynamic interactions between germ and Sertoli cells. The functional importance of sterol compounds in sperm production is further supported by the modulation of sterol composition in spermatozoal membranes during epididymal transit and in the female reproductive tract, which is a prerequisite for successful fertilization. However, the exact role of sterols in male reproduction is unknown. This review discusses sterol dynamics in sperm maturation and describes recent methodological advances that will help to illuminate the complexity of sperm formation and function. 相似文献
134.
Dunja Grbić‐Galić 《Geomicrobiology journal》2013,30(3-4):167-200
Fermentative and methanogenic bacteria have been found repeatedly as important members of microbial flora in anoxic zones of the subsurface—in pristine as well as in contaminated groundwater aquifers. These bacteria, which together with obligate proton reducers form complex methanogenic communities, are significant as decomposers of organic matter under conditions of exogenous electron acceptor depletion. Their metabolic activity has been demonstrated in laboratory microcosms derived from aquifer material, and also in the subsurface in situ. Methanogenic communities have been shown to transform numerous organic pollutants, or even to completely degrade these compounds with the production of carbon dioxide and methane. Depending on the chemical structure of the pollutant, such a compound can be used as an electron donor and a carbon/energy source for fermentative microorganisms (which is typically the case with highly reduced compounds); alternatively, a highly oxidized pollutant can be used as a potential electron acceptor or electron sink. This review addresses fermentative/methanogenic degradation of chlorinated and nonchlorinated aromatic hydrocarbons and phenols by subsurface microorganisms; for comparison, it briefly relates also other types of anaerobic transformations (under sulfate‐reducing, iron‐reducing, and denitrifying conditions). Furthermore, it outlines transformation pathways, those that are proposed as well as those that are already partially proved, for aromatic hydrocarbons and phenols under fermentative/methanogenic conditions; finally, it discusses the relevance of these processes to bioremediation of contaminated groundwater aquifers. 相似文献
135.
Despite the large number of publications on three‐helix protein folding, there is no study devoted to the influence of handedness on the rate of three‐helix protein folding. From the experimental studies, we make a conclusion that the left‐handed three‐helix proteins fold faster than the right‐handed ones. What may explain this difference? An important question arising in this paper is whether the modeling of protein folding can catch the difference between the protein folding rates of proteins with similar structures but with different folding mechanisms. To answer this question, the folding of eight three‐helix proteins (four right‐handed and four left‐handed), which are similar in size, was modeled using the Monte Carlo and dynamic programming methods. The studies allowed us to determine the orders of folding of the secondary‐structure elements in these domains and amino acid residues which are important for the folding. The obtained data are in good correlation with each other and with the experimental data. Structural analysis of these proteins demonstrated that the left‐handed domains have a lesser number of contacts per residue and a smaller radius of cross section than the right‐handed domains. This may be one of the explanations of the observed fact. The same tendency is observed for the large dataset consisting of 332 three‐helix proteins (238 right‐ and 94 left‐handed). From our analysis, we found that the left‐handed three‐helix proteins have some less‐dense packing that should result in faster folding for some proteins as compared to the case of right‐handed proteins.Proteins 2013; © 2013 Wiley Periodicals, Inc. 相似文献
136.
Theoretical Study of N‐Heterocyclic Carbenes‐Catalyzed Cascade Annulation of Benzodienones and Enals
Growing attention in developing new N‐heterocyclic carbene (NHC)‐mediated reactions involving homoenolate intermediates has prompted our interest in exploring the mechanistic details of the related reactions. In this work, we carried out a detailed theoretical study for the NHC‐catalyzed annulation reaction of cinnamaldehyde ( A ) and benzodi(enone) ( B ) in the presence of 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU). By performing density functional theory calculations, we show clearly the detailed reaction mechanism and rationalize the experimental observation. The reaction of A and B falls into two stages: the formation of homoenolate intermediate and the annulation of homoenolate with B . In the homoenolate formation stage, three possible paths are characterized. The pathway involving the DBU‐assisted 1,2‐proton transfer with a stepwise mechanism is kinetically more favorable, and the DBU‐assisted C1 proton departure is the rate‐determining step of the total reaction. The annulation of homoenolate with B involves four elementary steps. The conformational difference of homoenolate (cis and trans) leads to two slightly different reaction processes. In the total reaction, the process involving cis‐conformation of A is kinetically more feasible. This can be clearly understood through the frontier molecular orbital analysis and the electronic inductive effect. The calculated results are expected to offer valuable information for further design and development of NHC‐mediated reactions. Chirality 25:521‐528, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
137.
Hideo Shirafuji Shigeo Matsumura Ikuo Nogami Masahiko Yoneda 《Bioscience, biotechnology, and biochemistry》2013,77(12):3081-3084
To reveal the mechanism of reducing sugar-induced polymerization of proteins, monomeric lysozymes were isolated at various stages of storage from whole lysozyme (WL) being kept with glucose at 75% relative humidity and 50°C for up to 30 days, and their chemical properties were investigated and compared with the corresponding WL. The impairment of Lys, Arg, and Trp residues was observed in all the isolated monomeric lysozymes (IM) as well as in the WL.When the IM were stored for another 10 days without glucose, they polymerized and had an additional impairment of Arg and Lys but not Trp residues. All IM exhibited almost the same polymerization rate, but the sum of additional losses of Lys and Arg residues varied. The IM was also found to cross-link untreated lysozymes even in the absence of glucose.On basis of the results obtained hitherto, it is suggested that the glucose-induced polymerization of lysozymes proceeds through the following paths. At the first step, some bifunctional agents (BF), probably α-dicarbonyl compounds, generated from the reaction between ?-amino groups of lysine residues and glucose, attach to Arg, Lys, and Trp residues through one of their two functional sites. At the second step, some of those proteins with BF attached polymerize by binding of the other unoccupied functional site with the remaining Lys and Arg (not Trp) residues of the other protein molecules. The other of the proteins with BF attached polymerize through the combination between the other unoccupied functional sites themselves with no loss of amino acid residues. 相似文献
138.
甾体类药物是销售额仅次于抗生素的世界第二大类药物,不同的甾体药物分子结构均由甾体激素中间体衍生而来。甾体激素中间体的传统生产方法包括植物提取皂素法和化学全合成法,其对环境有害,反应产物结构不唯一且成本较高,不利于工业化生产。目前主要的生产工艺是利用微生物对特殊原料进行转化的半合成法,但会遇到微生物酶转化率低、发酵周期长等问题。合成生物学的出现为构建利用糖为唯一碳源生产甾体激素中间体的人工细胞提供了理论上的可行性和可靠的技术支持。重点综述了合成生物技术在甾体激素中间体生产中的应用,以有利于工业发酵的酿酒酵母、分枝杆菌等为底盘细胞,通过引入外源合成功能模块,实现胆甾醇、雄烯二酮等甾体激素中间体的生物合成,并对合成生物技术在医药生产方式转变中的应用进行了展望,以期推动甾体类药物生物制造技术的进步。 相似文献
139.
Combined quantum mechanics and molecular mechanics (QM/MM) calculations were carried out to characterize the reaction mechanism of the NS3 protease with its preferred substrate (NS5A/5B). The main purpose of this study was to locate the barrier states and intermediates along the distinguished coordinate path (DCP) involved in this process. These structures, and in particular the one corresponding to the first barrier state and intermediate (B1 and I1), could be a starting point for the synthesis of inhibitors of this protease, which could be used to treat hepatitis C. The two first steps of the reaction mechanism were studied, i.e., the acylation step and the breaking of the peptide bond. The first step takes place through a tetracoordinated intermediate, as suggested from previous works on other Serine proteases. The importance of the different amino acid residues was also considered (perturbation study where the MM charges of each residue were set to zero independently). The residues of the oxyanion hole were confirmed as the most important for the electrostatic stabilization of the tetracoordinate intermediate. Moreover, the role of other residues, e.g., Arg-155 and Asp-79, was also explained. 相似文献
140.
Exploring subdomain cooperativity in T4 lysozyme I: structural and energetic studies of a circular permutant and protein fragment
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Cellitti J Llinas M Echols N Shank EA Gillespie B Kwon E Crowder SM Dahlquist FW Alber T Marqusee S 《Protein science : a publication of the Protein Society》2007,16(5):842-851
Small proteins are generally observed to fold in an apparent two-state manner. Recently, however, more sensitive techniques have demonstrated that even seemingly single-domain proteins are actually made up of smaller subdomains. T4 lysozyme is one such protein. We explored the relative autonomy of its two individual subdomains and their contribution to the overall stability of T4 lysozyme by examining a circular permutation (CP13*) that relocates the N-terminal A-helix, creating subdomains that are contiguous in sequence. By determining the high-resolution structure of CP13* and characterizing its energy landscape using native state hydrogen exchange (NSHX), we show that connectivity between the subdomains is an important determinant of the energetic cooperativity but not structural integrity of the protein. The circular permutation results in a protein more easily able to populate a partially unfolded form in which the C-terminal subdomain is folded and the N-terminal subdomain is unfolded. We also created a fragment model of this intermediate and demonstrate using X-ray crystallography that its structure is identical to the corresponding residues in the full-length protein with the exception of a small network of hydrophobic interactions. In sum, we conclude that the C-terminal subdomain dominates the energetics of T4 lysozyme folding, and the A-helix serves an important role in coupling the two subdomains. 相似文献