115 research outputs found
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Structure and proposed mechanism of L-Ī±-glycerophosphate oxidase from Mycoplasma pneumoniae
The formation of hydrogen peroxide (HāOā) by the FAD-dependent Ī±-glycerophosphate oxidase (GlpO), is important for the pathogenesis of Streptococcus pneumoniae and Mycoplasma pneumoniae. The structurally known GlpO from Streptococcus sp. (SspGlpO) is similar to the pneumococcal protein (SpGlpO) and provides a guide for drug design against that target. However, M. pneumoniae GlpO (MpGlpO), having <20% sequence identity with structurally known GlpOs, appears to represent a second type of GlpO we designate as Type II GlpOs. Here, the recombinant His-tagged MpGlpO structure is described at ~2.5 Ć
resolution, solved by molecular replacement using as a search model the Bordetella pertussis protein 3253 (Bp3253) a protein of unknown function solved by structural genomics efforts. Recombinant MpGlpO is an active oxidase with a turnover number of ~580 minā»Ā¹ while Bp3253 showed no GlpO activity. No substantial differences exist between the oxidized and dithionite-reduced MpGlpO structures. Although, no liganded structures were determined, a comparison with the tartrate-bound Bp3253 structure and consideration of residue conservation patterns guided the construction of a model for Ī±-glycerophosphate (Glp) recognition and turnover by MpGlpO. The predicted binding mode also appears relevant for the type I GlpOs (such as SspGlpO) despite differences in substrate recognition residues, and it implicates a histidine conserved in type I and II Glp oxidases and dehydrogenases as the catalytic acid/base. This work provides a solid foundation for guiding further studies of the mitochondrial Glp dehydrogenases as well as for continued studies of M. pneumoniae and S. pneumoniae glycerol metabolism and the development of novel therapeutics targeting MpGlpO and SpGlpO.Keywords: drug design, flavoenzyme, protein evolution, GlpA, hydride transfe
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Kinetic Mechanism of L-Ī±-Glycerophosphate Oxidase from Mycoplasma pneumoniae
L-Ī±-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-Ī±-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (HāOā). The catalytic properties of the recombinant Hisā-GlpO from Mycoplasma pneumoniae (Hisā-MpGlpO) were investigated with transient and steady-state kinetics and ligand binding. The results indicate that the reaction mechanism of Hisā-MpGlpO follows a ping-pong model. Double-mixing stopped-flow experiments show that after flavin-mediate substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values of the individual rate constants and k [subscript]cat (4.2 sā»Ā¹ at 4 Ā°C) determined, in addition to the finding that HāOā can bind to the oxidized enzyme suggest that HāOā release is the rate-limiting step for the overall reaction. Results indicate that Hisā-MpGlpO contains mixed populations of fast and slow reacting species. Only the fast reacting species predominantly participates in turnovers. Different from other GlpO enzymes previously reported, Hisā-MpGlpO can catalyze the reverse reaction of reduced enzyme and DHAP. This result can be explained by the standard reduction potential value of Hisā-MpGlpO (-167 Ā± 1 mV), which is lower than those of GlpO from other species. We found that DL-glyceraldehyde 3-phosphate (GAP) can be used as a substrate in the Hisā-MpGlpO reaction, although it exhibited a ~100-fold lower k[subscript]cat value in comparison to the reaction of Glp. These results also imply the involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of Hisā-MpGlpO reported here should be useful for future studies of drug development against Mycoplasma pneumoniae infection
Turnover-Dependent Covalent Inactivation of Staphylococcus aureus Coenzyme A-Disulfide Reductase by Coenzyme A-Mimetics: Mechanistic and Structural Insights
Disruption of the unusual thiol-based redox homeostasis mechanisms in Staphylococcus aureus represents a unique opportunity to identify new metabolic processes, and new targets for intervention. Targeting uncommon aspects of CoASH biosynthetic and redox functions in S. aureus, the antibiotic CJ-15,801 has recently been demonstrated to be an antimetabolite of the CoASH biosynthetic pathway in this organism; CoAS-mimetics containing Ī±,Ī²-unsaturated sulfone and carboxyl moieties have also been exploited as irreversible inhibitors of S. aureus coenzyme A-disulfide reductase (SaCoADR). In this work we have determined the crystal structures of three of these covalent SaCoADR-inhibitor complexes, prepared by inactivation of wild-type enzyme during turnover. The structures reveal the covalent linkage between the active-site Cys43-SĪ³ and CĪ² of the vinyl sulfone or carboxyl moiety. The full occupancy of two inhibitor molecules per enzyme dimer, together with kinetic analyses of the wild-type/C43S heterodimer, indicates that half-sites-reactivity is not a factor during normal catalytic turnover. Further, we provide the structures of SaCoADR active-site mutants; in particular, Tyr419ā²-OH plays dramatic roles in directing intramolecular reduction of the Cys43-SSCoA redox center, in the redox asymmetry observed for the two FAD per dimer in NADPH titrations, and in catalysis. The two conformations observed for the Ser43 side chain in the C43S mutant structure lend support to a conformational switch for Cys43-SĪ³ during its catalytic Cys43-SSCoA/Cys43-SH redox cycle. Finally, the structures of the three inhibitor complexes provide a framework for design of more effective inhibitors with therapeutic potential against several major bacterial pathogens
Characterization of the N -Acetyl-Ī±- d -glucosaminyl l -Malate Synthase and Deacetylase Functions for Bacillithiol Biosynthesis in Bacillus anthracis ,
Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular-weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce Ī±-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (āGlcNAc-malate) and the BaBshB deacetylase (āGlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Ć
resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other Ī±-hydroxyacids, as acceptor substrate. In sharp contrast to the structures reported for the GlcNAcā1-D-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon site hybridization study as required for growth, sporulation, or germination, suggesting that the biosynthesis of BSH could represent a target for development of novel antimicrobials with broad spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work
Sushi in the United States, 1945-1970
Sushi first achieved widespread popularity in the United States in
the mid-1960s. Many accounts of sushiās US establishment foreground
the role of a small number of key actors, yet underplay
the role of a complex web of large-scale factors that provided the
context in which sushi was able to flourish. This article critically
reviews existing literature, arguing that sushiās US popularity
arose from contingent, long-term, and gradual processes. It examines
US newspaper accounts of sushi during 1945ā1970, which
suggest the discursive context for US acceptance of sushi was
considerably more propitious than generally acknowledged.
Using California as a case study, the analysis also explains
conducive social and material factors, and directs attention to
the interplay of supply- and demand-side forces in the favorable
positioning of this ānewā food. The article argues that the US
establishment of sushi can be understood as part of broader
public acceptance of Japanese cuisine
Cell signalling by reactive lipid species: new concepts and molecular mechanisms
The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the ācovalent advantageā. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway
Enzymatic oligomerization and polymerization of arylamines: state of the art and perspectives
The literature concerning the oxidative oligomerization and polymerization of various arylamines, e.g., aniline, substituted anilines, aminonaphthalene and its derivatives, catalyzed by oxidoreductases, such as laccases and peroxidases, in aqueous, organic, and mixed aqueous organic monophasic or biphasic media, is reviewed. An overview of template-free as well as template-assisted enzymatic syntheses of oligomers and polymers of arylamines is given. Special attention is paid to mechanistic aspects of these biocatalytic processes. Because of the nontoxicity of oxidoreductases and their high catalytic efficiency, as well as high selectivity of enzymatic oligomerizations/polymerizations under mild conditions-using mainly water as a solvent and often resulting in minimal byproduct formation-enzymatic oligomerizations and polymerizations of arylamines are environmentally friendly and significantly contribute to a "green'' chemistry of conducting and redox-active oligomers and polymers. Current and potential future applications of enzymatic polymerization processes and enzymatically synthesized oligo/polyarylamines are discussed
Oxygen Reactivity of an NADH Oxidase C42S Mutant:Ā Evidence for a C(4a)-Peroxyflavin Intermediate and a Rate-Limiting Conformational Change ā
4-Thioflavins as active site probes of flavoproteins : reactions with sulfite
4-Thioflavins react with sulfite under aerobic conditions to yield highly fluorescent products with absorption maxima aroun 410 nm. These producths have been identified as 4-hydroxy-4-sulfonylflavins, and have been shown to arise from a series of reactions following the O2-dependent reoxidation of an intermediate with absorption maxima at 363 and 465 nm. Under anaerobic conditions, the same intermediate is formed, but decays to a 350 nm absorbing species, which is probably the N(5)-sulfite adduct of 4-thioflavin. A plausible mechanism is described for the formation of the derivatives, and several of their chemical and physical properties are described. Distinctly different results between different proteins are obtained when sulfite reacts with enzyme-bound 4-thioflavins. 4-Thio-FAD-D-amino acid oxidase and 4-thio-FMN-lactate oxidase react rapidly to yield the N(5)-sulfite adducts, as occurs with the native enzymes. 4-Thio- FAD-p-hydroxybenzoate hydroxylase reacts slowly in a manner paralleling the reaction with the free 4- thioflavins
4-Thioflavins as active site probes of flavoproteins : general properties
4-Thioflavins (oxygen at position 4 replaced by sulfur) have been studied as potential active site probes of flavoproteins. They react readily with thiol reagents,with large spectral changes, which should be useful for testing the accessibility of the flavin 4-position in flavoproteins. They have an oxidation-reduction potential at pH 7 of -0.055 V, approximately 0.15 V higher than that of native flavins. The spectral characteristics in the fully reduced state show two clear absorption bands, dependent on the ionization state (pK = 4.5). The lowest energy band of the neutral dihydroflavin has a maximum at ~485 nm while that of the anion is ~425 nm. This should be useful in defining the ionization state of the reduced flavin in flavoproteins. The spectral characteristics of the semiquinoid forms of 4-thioflavins have been determined bound to the apoproteins of flavodoxin and D-amino acid oxidase. The neutral radical has an absorption maximum at 730 nm, while the anion radical has an unusually sharp peak at 415 nm. The reduced forms of 4-thioflavins, free and enzyme bound, react with O2 to regenerate oxidized 4-thioflavin. Reduced 4-thio-FAD p-hydroxybenzoate hydroxylase, however, in its reaction with 02, undergoes a substantial conversion to the native FAD-enzyme. 4-Thioflavins are unusually susceptible to attack by nucleophiles such as hydroxylamine and amines to form the respective 4-hydroxyimino- and 4-aminoflavins, offering the possibility of forming stable covalent flavin-protein linkages with suitably positioned protein residues. Thiols also react with 4-thioflavins, promoting their conversion to the normal (4- oxo) flavin coenzymes. Such reactivity has been found with the apoenzymes of glucose oxidase and lactate oxidase, providing evidence for a thiol residue in the active site of these enzymes
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