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1.
The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K(+) site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25°C. Using UV-visible spectrophotometry, we show that the removal of K(+) binding site causes substantial changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I were not observed in the K(+) site double mutants in the presence of H(2)O(2). Similarly electron donor oxidation rate was two orders less for the K(+) site double mutants compared to the wild type. These data show that K(+) functions in preserving the protein structure in the heme surroundings as well as the spin state of the heme iron, in favor of the enzymatically active form of LmP. 相似文献
2.
Yolanda Pérez-Pertejo Rosa M Reguera Hector Villa Carlos García-Estrada Rafael Bala?a-Fouce Maria A Pajares David Ordó?ez 《European journal of biochemistry》2003,270(1):28-35
Methionine adenosyltransferase (MAT, EC 2.5.1.6)-mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities, AdoMet synthesis and tripolyphosphate hydrolysis, can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. The present report describes the cloning and subsequent functional refolding, using a bacterial expression system, of the MAT gene (GenBank accession number AF179714) from Leishmania donovani, the etiological agent of visceral leishmaniasis. The absolute need to include a sulfhydryl-protection reagent in the refolding buffer for this protein, in conjunction with the rapid inactivation of the functionally refolded protein by N-ethylmaleimide, suggests the presence of crucial cysteine residues in the primary structure of the MAT protein. The seven cysteines in L. donovani MAT were mutated to their isosterical amino acid, serine. The C22S, C44S, C92S and C305S mutants showed a drastic loss of AdoMet synthesis activity compared to the wild type, and the C33S and C47S mutants retained a mere 12% of wild-type MAT activity. C106S mutant activity and kinetics remained unchanged with respect to the wild-type. Cysteine substitutions also modified PPPi cleavage and AdoMet induction. The C22S, C44S and C305S mutants lacked in tripolyphosphatase activity altogether, whereas C33S, C47S and C92S retained low but detectable activity. The behavior of the C92S mutant was notable: its inability to synthesize AdoMet combined with its retention of tripolyphosphatase activity appear to be indicative of the specific involvement of the respective residue in the first step of the MAT reaction. 相似文献
3.
Leishmania major peroxidase (LmP) exhibits both ascorbate and cytochrome c peroxidase activities. Our previous results illustrated that LmP has a much higher activity against horse heart cytochrome c than ascorbate, suggesting that cytochrome c may be the biologically important substrate. To elucidate the biological function of LmP, we have recombinantly expressed, purified, and determined the 2.08 ? crystal structure of L. major cytochrome c (LmCytc). Like other types of cytochrome c, LmCytc has an electropositive surface surrounding the exposed heme edge that serves as the site of docking with redox partners. Kinetic assays performed with LmCytc and LmP show that LmCytc is a much better substrate for LmP than horse heart cytochrome c. Furthermore, unlike the well-studied yeast system, the reaction follows classic Michaelis-Menten kinetics and is sensitive to an increasing ionic strength. Using the yeast cocrystal as a control, protein-protein docking was performed using Rosetta to develop a model for the binding of LmP and LmCytc. These results suggest that the biological function of LmP is to act as a cytochrome c peroxidase. 相似文献
4.
We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H(2)O(2) with concurrent evolution of O(2). This pseudocatalatic degradation of H(2)O(2) to O(2) is solely dependent on ascorbate and is blocked by a spin trap, alpha-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H(2)O with subsequent regeneration of ascorbate by the reduction of MDA with H(2)O(2) evolving O(2) through the intermediate formation of O(2)(-). Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN(-) in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN(-). Further kinetic studies indicate that SCN(-) itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN(-) blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H(2)O(2) to O(2). Binding studies by optical difference spectroscopy indicate that SCN(-) binds LmAPX (Kd = 100 +/- 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN(-) acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity. 相似文献
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6.
Balog EM Norton LE Thomas DD Fruen BR 《American journal of physiology. Heart and circulatory physiology》2006,290(2):H794-H799
Calmodulin (CaM) binds to the cardiac ryanodine receptor Ca2+ release channel (RyR2) with high affinity and may act as a regulatory channel subunit. Here we determine the role of CaM Met residues in the productive association of CaM with RyR2, as assessed via determinations of [3H]ryanodine and [35S]CaM binding to cardiac muscle sarcoplasmic reticulum (SR) vesicles. Oxidation of all nine CaM Met residues abolished the productive association of CaM with RyR2. Substitution of the COOH-terminal Mets of CaM with Leu decreased the extent of CaM inhibition of cardiac SR (CSR) vesicle [3H]ryanodine binding. In contrast, replacing the NH2-terminal Met of CaM with Leu increased the concentration of CaM required to inhibit CSR [3H]ryanodine binding but did not alter the extent of inhibition. Site-specific substitution of individual CaM Met residues with Gln demonstrated that Met124 was required for both high-affinity CaM binding to RyR2 and for maximal CaM inhibition. These results thus identify a Met residue critical for the productive association of CaM with RyR2 channels. 相似文献
7.
In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified alpha, beta, and gamma subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg(-1)). Kinetic studies suggest a ternary complex mechanism, with Km values of 140 mum for trypanothione and 7.4 mm for cumene hydroperoxide and kcat=25 s(-1). Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation. 相似文献
8.
Jasion VS Polanco JA Meharenna YT Li H Poulos TL 《The Journal of biological chemistry》2011,286(28):24608-24615
The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX). One common feature is a key Trp residue, Trp(208) in LmP and Trp(191) in CCP, that is situated adjacent to the proximal His heme ligand in CCP, APX, and LmP. In CCP, Trp(191) forms a stable cationic radical after reaction with H(2)O(2) to form Compound I; in APX, the radical is located on the porphyrin ring. In order to clarify the role of Trp(208) in LmP and to further probe peroxidase structure-function relationships, we have determined the crystal structure of LmP and have studied the role of Trp(208) using electron paramagnetic resonance spectroscopy (EPR), mutagenesis, and enzyme kinetics. Both CCP and LmP have an extended section of β structure near Trp(191) and Trp(208), respectively, which is absent in APX. This region provides stability to the Trp(191) radical in CCP. EPR of LmP Compound I exhibits an intense and stable signal similar to CCP Compound I. In the LmP W208F mutant, this signal disappears, indicating that Trp(208) forms a stable cationic radical. In LmP conversion of the Cys(197) to Thr significantly weakens the Compound I EPR signal and dramatically lowers enzyme activity. These results further support the view that modulation of the local electrostatic environment controls the stability of the Trp radical in peroxidases. Our results also suggest that the biological role of LmP is to function as a cytochrome c peroxidase. 相似文献
9.
Heme peroxidases are a class of multifunctional redox-active proteins found in all organisms. We recently cloned, expressed, and characterized an ascorbate peroxidase from Leishmania major (LmAPX) that was capable of detoxifying hydrogen peroxide. Localization studies using green fluorescent protein fusions revealed that LmAPX was localized within the mitochondria by its N-terminal signal sequence. Subcellular fractionation analysis of the cell homogenate by the Percoll density-gradient method and subsequent Western blot analysis with anti-LmAPX antibody further confirmed the mitochondrial localization of mature LmAPX. Submitochondrial fractionation analysis showed that the mature enzyme (~3.6 kDa shorter than the theoretical value of the whole gene) was present in the intermembrane space side of the inner membrane. Moreover, expression of the LmAPX gene was increased by treatment with exogenous H(2)O(2), indicating that LmAPX was induced by oxidative stress. To investigate the biological role of LmAPX we generated Leishmania cells overexpressing LmAPX in the mitochondria. Flow-cytometric analysis, thin-layer chromatography, and IC(50) measurements suggested that overexpression of LmAPX caused depletion of the mitochondrial ROS burden and conferred a protection against mitochondrial cardiolipin oxidation and increased tolerance to H(2)O(2). These results suggest that the single-copy LmAPX gene plays a protective role against oxidative damage. 相似文献
10.
Oxidation of methionine residues in lutropin 总被引:1,自引:0,他引:1
Bovine lutropin and its subunits were submitted to oxidation by sodium periodate or chloramine T. Methionine residues were easily oxidized but partial destruction of fucose was observed. After oxidation treatment most of the lutropin exhibits the same elution volume in gel filtration as the native hormone. Sucrose gradient sedimentation or gel filtration experiments show however that the oxidation of isolated subunits is accompanied by aggregation of conformational changes even in the case of porcine beta subunit which contains only one methionine residue, unit and intact alpha subunit exhibit very low residual activity. 相似文献
11.
Esaú E. Rodríguez Trinidad Arcos-López Lidia G. Trujano-Ortiz Claudio O. Fernández Felipe J. González Alberto Vela Liliana Quintanar 《Journal of biological inorganic chemistry》2016,21(5-6):691-702
Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson’s disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1–6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1–6) fragment, and in the redox activity of the Cu–AS(1–6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)–AS(1–6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu–AS(1–6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu–AS complex. 相似文献
12.
Folylpolyglutamates in Leishmania major 总被引:3,自引:0,他引:3
D V Santi P Nolan B Shane 《Biochemical and biophysical research communications》1987,146(3):1089-1092
The intracellular folates of the protozoan parasite Leishmania major have been examined. About 95% of the exogenous [3H]folate accumulated by the protozoan is metabolized to polyglutamate conjugates within 65 hr, and the intracellular folates are about forty-fold concentrated over the folate in the medium. The predominant metabolite of folic acid is the pentaglutamate conjugate (85%), with lessor amounts of the tetraglutamate (approximately 9%) and hexaglutamate (approximately 3%), and trace (less than 2.5%) amounts of di-, tri- and hepta-glutamate conjugates. Chromatographic properties of the products indicate that the conjugates are linked through the gamma-carboxyl groups. The folylpolyglutamate distribution in Leishmania is similar to that found in mammalian tissues. 相似文献
13.
Ethan J. Walker John Q. Bettinger Kevin A. Welle Jennifer R. Hryhorenko Adrian M. Molina Vargas Mitchell R. OConnell Sina Ghaemmaghami 《The Journal of biological chemistry》2022,298(5)
The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability. 相似文献
14.
Biochemistry of methionine sulfoxide residues in proteins 总被引:6,自引:0,他引:6
The oxidation of methionine to methionine sulfoxide constitutes one of the many post-translational modifications that proteins undergo. This non-enzymatic reaction has been shown to occur both in vivo and in vitro, and has been associated with the loss of biological activity in a wide variety of proteins and peptides. The presence of methionine sulfoxide residues in proteins is implicated in a variety of pathological conditions. An enzyme that is present in all organisms tested specifically catalyzes the reduction of the methionine sulfoxide residues in proteins. The physiological reductant for this enzyme appears to be thioredoxin. 相似文献
15.
Lüersen K 《FEBS letters》2005,579(24):5347-5352
Thialysine N(epsilon)-acetyltransferases and spermidine/spermine N-acetyltransferases (SSAT) are closely related members of the GCN5-related N-acetyltransferase superfamily. Accordingly, a putative orthologue from the human protozoan parasite Leishmania major exhibits an almost equal similarity to human SSAT and thialysine N(epsilon)-acetyltransferase. Characterisation of the recombinantly expressed L. major protein indicated that it represents a thialysine N(epsilon)-acetyltransferase, preferring thialysine (S-aminoethyl-l-cysteine) and structurally related amino acids as acceptor molecules. The known thialysine N(epsilon)-acetyltransferases contain five conserved amino acid residues that are replaced in SSAT sequences. Kinetic analyses of the respective recombinant mutant proteins suggest that Ser(82) and Thr(83) of L. major thialysine N(epsilon)-acetyltransferase are key residues for acceptor binding. In addition, the conserved Leu(130) is tentatively involved in specific interaction with the sulphur-containing side chain of thialysine. The presence of these three amino acid residues is suggested to be a means by which thialysine N(epsilon)-acetyltransferases can be distinguished from SSAT sequences. 相似文献
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17.
Drummelsmith J Girard I Trudel N Ouellette M 《The Journal of biological chemistry》2004,279(32):33273-33280
Leishmania is a trypanosomatid parasite causing serious disease and displaying resistance to various drugs. Here, we present comparative proteomic analyses of Leishmania major parasites that have been either shocked with or selected in vitro for high level resistance to the model antifolate drug methotrexate. Numerous differentially expressed proteins were identified by these experiments. Some were associated with the stress response, whereas others were found to be overexpressed due to genetic linkage to primary resistance mediators present on DNA amplicons. Several proteins not previously associated with resistance were also identified. The role of one of these, methionine adenosyltransferase, was confirmed by gene transfection and metabolite analysis. After a single exposure to low levels of methotrexate, L. major methionine adenosyltransferase transfectants could grow at high concentrations of the drug. Methotrexate resistance was also correlated to increased cellular S-adenosylmethionine levels. The folate and S-adenosylmethionine regeneration pathways are intimately connected, which may provide a basis for this novel resistance phenotype. This thorough comparative proteomic analysis highlights the variety of responses required for drug resistance to be achieved. 相似文献
18.
Mattie C. Pawlowic Fong‐fu Hsu Samrat Moitra Neha Biyani Kai Zhang 《Molecular microbiology》2016,101(2):238-249
Ethanolamine glycerophospholipids are ubiquitous cell membrane components. Trypanosomatid parasites of the genus Leishmania synthesize the majority of their ethanolamine glycerophospholipids as 1‐O‐alk‐1′‐enyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamine or plasmenylethanolamine (PME) through the Kennedy pathway. PME is a subtype of ether phospholipids also known as ethanolamine plasmalogen whose functions are not well characterized. In this study, we investigated the role of PME synthesis in Leishmania major through the characterization of an ethanolamine phosphotransferase (EPT) mutant. EPT‐null parasites are largely devoid of PME and fully viable in regular medium but fail to proliferate in the absence of fetal bovine serum. They exhibit significant abnormalities in the synthesis and localization of GPI‐anchored surface molecules. EPT‐null mutants also show attenuated virulence in BALB/c mice. Furthermore, in addition to PME synthesis, ethanolamine also contributes to the production of phosphatidylcholine, the most abundant class of lipids in Leishmania. Together, these findings suggest that ethanolamine production is likely required for Leishmania promastigotes to generate bulk phospholipids, to handle stress, and to control the expression of membrane bound virulence factors. 相似文献
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20.
Azurin from has been treated with bromoacetate at low pH to alkylate methionine residues. Two classes of methionine side chains are observed as a result of these reactions — four of the six methionines are reactive at pH 4, whereas all six are reactive at pH 3.2. The product containing four alkylated methionines maintains a significant portion of the blue color and spectroscopic characteristics of the native protein. The product which has been fully modified at the methionine residues, on the other hand, has lost all blue color and appears to be largely in a random coil form. 相似文献