Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NO synthase (NOS) is a unique P-450-type enzyme containing both a reductase and a heme domain on a single polypeptide. We show that ebselen [Ebs, 2-phenyl-1,2-benzisoselenazol-3-(2H) one], a nontoxic selenoorganic compound known to break a cysteine thiolate/Fe bond of some of P-450 enzymes, is a relatively selective inhibitor of endothelial isoform of NOS. In rings of rabbit aorta, Ebs irreversibly blocked both the basal as well as acetylcholine- or calcium ionophore A23187-stimulated release of nitric oxide with an IC50 of 6 microM. In homogenates of bovine aortic endothelial cells, Ebs inhibited the activity of NOS, assayed by monitoring conversion of L-[2,3-3H]arginine to L-[2,3-3H]citrulline, with an IC50 of 8.5 microM. The inhibitory action of Ebs was prevented by glutathione, N-acetyl-L-cysteine or dithiothreitol (30-500 microM). The prevention by thiols of Ebs-induced inhibition of NOS suggests that these are competing with a thiol group of NOS that is essential for the catalytic activity of the enzyme. The consequence of the presence of thiols is the "trapping" of Ebs in the form of inactive selenyl sulfides. Consistent with the proposed mechanism of action of Ebs is lack of activity of diselenide of Ebs, which also demonstrates that the action of Ebs is independent of its glutathione peroxidase-like activity. In comparison to endothelial preparations, IC50 values of Ebs for inhibition of soluble isoforms of NOS present in homogenates of porcine cerebellum and of spleens obtained from lipopolysaccharide-treated rats were more than 30-fold higher.
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PMID:Inhibition of endothelial nitric oxide synthase by ebselen. Prevention by thiols suggests the inactivation by ebselen of a critical thiol essential for the catalytic activity of nitric oxide synthase. 750 26

CDP-6-deoxy-delta 3,4-glucoseen reductase (E3), which catalyzes the reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis, has been expressed at high level in Escherichia coli (670 times over the wild-type strain). This flavoenzyme, which also contains one plant ferredoxin type [2Fe-2S] cluster, was inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide. In both cases the inactivation followed a pseudo first order kinetics. The second order rate constant for the reaction of DTNB with E3 was 0.25 mM-1 min-1 at 20 degrees C, pH 8.0. Detailed characterization of the inactivated enzyme showed that neither the flavin nor the [2Fe-2S] cluster was altered during inactivation. Since this inactivation was reversible by treating the inactivated enzyme with 1 mM D,L-dithiothreitol (DTT), it was concluded that only cysteine residues were modified during inactivation. Analysis of the inactivation using the method developed by Tsou revealed that two cysteines react with DTNB at similar rates and modification of either one is enough to impair E3's activity. Tryptic digestion of E3 labeled with N-ethyl[2,3-14C]maleimide, followed by fractionation of the digest by high performance liquid chromatography, gave two labeled peptides, both of which were separately isolated as a pair of interconvertible diastereoisomers. Sequence analysis of these labeled peptides allowed the identification of Cys-75 and Cys-296 as the reactive cysteine residues. Interestingly, the C75S and C296S mutant proteins exhibit identical physical and comparable catalytic properties as the wild-type enzyme. Since Cys-296 is a conserved residue in the NAD(P) binding domain of enzymes belonging to the same class, this residue may be involved in stabilizing the charge-transfer complex between E3 and NADH, thus facilitating hydride transfer from the nicotinamide nucleotide to flavin. A chemically modified Cys-75 which is immediately adjacent to the [2Fe-2S] center in E3 may prevent the proper juxtaposition of the redox centers and thus impede electron transfer leading to enzyme inactivation. These results may be useful for placing constraints on the peptide folding comprising the active site of E3 for electron transfer between NADH, FAD, and the [2Fe-2S] center.
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PMID:Mechanistic studies on CDP-6-deoxy-delta 3,4-glucoseen reductase: the role of cysteine residues in catalysis as probed by chemical modification and site-directed mutagenesis. 770 27

To better understand the extrarenal production of active vitamin D metabolites by cells of the monocyte/macrophage lineage, we investigated the 25-hydroxyvitamin D (25OHD)-1-hydroxylation reaction in the v-myc-transformed chick myelomonocytic cell line HD-11; the 1-hydroxylation reaction in this cell line has a high affinity for 25-hydroxylated vitamin D substrates, is localized to mitochondria, and is associated with cytochrome P450 activity. In this study we demonstrated that the HD-11 cell 1-hydroxylation reaction in vitro is not affected by the majority of extracellular regulatory factors that modulate expression of the renal 25OHD-1-hydroxylase in vivo. A 50% increase in extracellular calcium and phosphate concentrations, physiological inhibitory events for renal 1,25-dihydroxyvitamin D [1,25-(OH)2D] synthesis, did not decrease basal expression of the HD-11 cell 1-hydroxylation reaction, nor did a 50% decrease in extracellular calcium and phosphate concentrations, stimulatory signals for the 1-hydroxylase in vivo, increase 1,25-(OH)2D3 synthesis in vitro. Receptor-saturating concentrations of PTH and PTH-related peptide were similarly without effect. In contrast, the HD-11 1-hydroxylation reaction was significantly stimulated in a dose-dependent fashion by the macrophage stimulatory agents lipopolysaccharide [P < 0.001 at a maximum effective concentration (EC100) of 25 micrograms/ml] and interferon-gamma (P < 0.001 at EC100 of 1000 IU/ml) and by insulin-like growth factor-I (P < 0.01 at EC100 of 15 nM) with the rank order of stimulation being interferon-gamma > lipopolysaccharide > insulin-like growth factor-I. Dexamethasone (> or = 10 nM) and the cytochrome P450 inhibitors (EC100, 20 microM), ketoconazole, clotrimazole, and menadione, all significantly inhibited the HD-11 cell 1-hydroxylation reaction. The naphthoquinone menadione, which blocks electron transfer to the P450-associated enzyme, was the most effective inhibitor of the reaction in both intact cells (3 +/- 1% of basal expression; P < or = 0.002) and after reconstitution of HD-11 cell mitochondrial extracts with a ferredoxin, reductase, O2, and NADPH (5 +/- 1% of basal; P < or = 0.02). We have also shown that 1,25-(OH)2D3 produced from substrate 25OHD3 appears to exert an endogenous (intracrine) inhibitory effect on HD-11 cell growth; incubation of HD-11 cells with a concentration of ketoconazole (10 microM) known to reduce 1,25-(OH)2D3 production by roughly 50% restored 50% of the growth deficit induced by 1,25-(OH)2D3 (EC100, 100 nM).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulated production and intracrine action of 1,25-dihydroxyvitamin D3 in the chick myelomonocytic cell line HD-11. 819 84

The highly regulated enzyme HMG-CoA reductase generates mevalonate, the precursor of a complex series of isoprenoids that posttranslationally modify (isoprenylate) certain proteins (e.g., the low-molecular-weight GTP-binding proteins) or that are incorporated into cholesterol and other end products. We recently reported that isoprenoids are required for NADPH oxidase activity in granulocytes via LMW GTP-binding protein isoprenylation. In this study, we evaluated the effects of isoprenoid depletion on the expression of proinflammatory genes in human monocytic THP-1 cells. We selected conditions under which pretreatment for 24 h with isoprenoid synthesis inhibitors (HMG-CoA reductase inhibitor lovastatin or compactin at 10 microM) did not compromise cell viability but markedly suppressed H2O2 generation. Under these conditions interleukin-8 (IL-8) production was attenuated (by 50-90%) in response to lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, and phorbol myristate acetate. Coincubation of reductase inhibitor-treated cells with mevalonate prevented the attenuation of IL-8 production by reductase inhibitors. The effects of isoprenoid depletion on cytokine production were selective: IL-1 beta generation was not inhibited but the production of IL-6 and IL-8 was concomitantly suppressed. IL-8 induction was suppressed at least in part through attenuation of the increase in mRNA in stimulated cells. We conclude that isoprenoid generation through the mevalonate pathway is a requirement for IL-8 induction by activated monocytic cells in vitro. Isoprenylation inhibitors have the potential to alter monocyte proinflammatory function.
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PMID:Role of the mevalonate pathway of isoprenoid synthesis in IL-8 generation by activated monocytic cells. 819 1

The CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) is a NADH-dependent enzyme which catalyzes the key reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis. This highly purified enzyme is also a NADH oxidase capable of mediating the direct electron transfer from NADH to O2, forming H2O2. While previous work showed that E3 contains no common cofactor, one FAD and one plant ferredoxin type [2Fe-2S] center were found in this study to be associated with each molecule of E3. The iron-sulfur center is essential for E3 activity since bleaching of the [2Fe-2S] center leads to inactive enzyme. These results suggest that E3 employs a short electron-transport chain composed of both FAD and the iron-sulfur center to shuttle electrons from NADH to its acceptor. The order of electron flow, as indicated by EPR measurement with partially reduced E3, starts with hydride reduction of FAD by NADH. The iron-sulfur cluster, receiving electrons one at a time from the reduced flavin, relays the reducing equivalents via another iron-sulfur center in the active site of E1 to its final acceptor, the E1-bound PMP-glucoseen adduct. The participation of a one-electron-carrying iron-sulfur center in this reduction is advantageous since both electrons are dispatched from the same redox state of the prosthetic group, allowing electrons of equal energy to be delivered to the final acceptor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cofactor characterization and mechanistic studies of CDP-6-deoxy-delta 3,4-glucoseen reductase: exploration into a novel enzymatic C-O bond cleavage event. 821 67

The 3,6-dideoxyhexoses, usually confined to the cell wall lipopolysaccharide of gram-negative bacteria, are essential to serological specificity and are formed via a complex biosynthetic pathway beginning with CDP-D-hexoses. In particular, the biosynthesis of CDP-ascarylose, one of the naturally occurring 3,6-dideoxyhexoses, consists of five enzymatic steps, with CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) participating as the key enzyme in this catalysis. This enzyme has been previously purified from Yersinia pseudotuberculosis by an unusual procedure (protocol I) including a trypsin digestion step (O. Han, V.P. Miller, and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the cloned gene showed disparity with the expected gene characteristics, and upon expression, the resulting gene product exhibited no E3 activity. These findings strongly suggested that the protein isolated by protocol I may have been misidentified as E3. A reinvestigation of the purification protocol produced a new and improved procedure (protocol II) consisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Sephadex G-100 chromatography, which efficiently yielded a new homogeneous enzyme composed of a single polypeptide with a molecular weight of 39,000. This highly purified protein had a specific activity nearly 8,000-fold higher than that of cell lysates, and more importantly, the corresponding gene (ascD) was found to be part of the ascarylose biosynthetic cluster. Presented are the identification and confirmation of the E3 gene through cloning and overexpression and the culminating purification and unambiguous assignment of homogeneous E3. The nucleotide and translated amino acid sequences of the genuine E3 are also presented.
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PMID:CDP-6-deoxy-delta 3,4-glucoseen reductase from Yersinia pseudotuberculosis: enzyme purification and characterization of the cloned gene. 828 41

The first four genes (rfbA,B,D,E) of the rfb region of Vibrio cholerae O1 are predicted to encode the enzymes required for the biosynthesis of perosamine, which constitutes the backbone structure of the O-antigen of the lipopolysaccharide. Based on homology to known proteins/protein families, the following functions are predicted: RfbA, phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase; RfbB, phosphomanno-mutase; RfbD, oxido reductase and RfbE, perosamine synthetase (amino-transferase). Thus, perosamine is synthesized from fructose 6-phosphate via the intermediates mannose 6-phosphate by RfbA, to mannose 1-phosphate by RfbB, to GDP-mannose by RfbA, to GDP-4-keto-6-dideoxymannose by RfbD and to GDP-perosamine by RfbE. This final product would then serve as the substrate for the addition of the tetronate, which could then be polymerized into the O-antigen for transfer to the lipid A plus core oligosaccharide and export to the cell surface. The organization of these genes are such that one would expect them to be translationally coupled as part of the rfb operon. However, the absence of readily detectable promoter sequences suggests low levels of transcription, in line with other studies. The nucleotide sequence of these genes is absolutely conserved in the two isolates 569B (classical, Inaba) and O17 (El Tor, Ogawa) which were expected to show maximal sequence variation. This suggests very tight constraints on the micro-evolution within these sequences.
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PMID:A putative pathway for perosamine biosynthesis is the first function encoded within the rfb region of Vibrio cholerae O1. 852 91

Bactericidal/permeability-increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram-negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell-surface damage by BPI, two-dimensional gel electrophoresis and N-terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the beta-subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11-fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl-acyl carrier protein reductase, and the heat-shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct-repeat motif is present in the regulatory regions of several BPI-inducible genes, including the bipA gene. Only one of the BPI-responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell-surface component.
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PMID:Salmonella typhimurium responses to a bactericidal protein from human neutrophils. 855 71

The conversion of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideoxy-D-glucose is a key step in biosynthesis of ascarylose, the terminal dideoxyhexose of the O-antigen tetrasaccharide of the lipopolysaccharide from Yersinia pseudotuberculosis V. This transformation is catalyzed by two enzymes: CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which contains a pyridoxamine and a [2Fe-2S] center, and an NADH-dependent CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), which contains both an FAD and a [2Fe-2S] center. E1 reacts to form a Schiff base with CDP-4-keto-6-deoxy-D-glucose and catalyzes the elimination of the hydroxyl at position 3 of the glucose moiety, resulting in the formation of a covalently bound CDP-6-deoxy-delta(3,4)-glucoseen intermediate. E3 transfers electrons from NADH to E1, which uses these to reduce the delta(3,4)-glucoseen bond to produce CDP-4-keto-3,6-dideoxy-D-glucose. In this work, we have investigated the reductive half-reaction of E3 using both single wavelength and diode array stopped flow absorbance spectroscopy. We find that NADH binds to both oxidized (Kd = 52.5 +/- 2 microM) and two-electron-reduced (Kd = 12.1 +/- 1 microM) forms of E3. Hydride transfer from NADH to the FAD moiety occurs at 107.5 +/- 3 s-1 and exhibits a 10-fold deuterium isotope effect when (4R)-[2H]NADH is substituted for NADH. Following the hydride transfer reaction, NAD+ is released at 42.5 +/- 1 s-1 and electron transfer from the reduced FAD to the [2Fe-2S] center occurs rapidly. The extent of the intramolecular electron transfer reaction is pH-dependent with a pKa of 7.3 +/- 0.1, which may represent the ionization state of the N-1 position of the FAD hydroquinone of E3. Finally, E3 is converted to the three-electron-reduced state in a slow disproportionation reaction that consumes NADH: The [2Fe-2S] center of E3 was selectively disassembled by titration with mersalyl to give E3(apoFeS). The properties of this form of the enzyme are compared to those of the holoenzyme. Similarities and differences of the reductive half-reactions of E3 and related iron-sulfur flavoenzymes are discussed.
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PMID:Kinetics of the reductive half-reaction of the iron-sulfur flavoenzyme CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase. 867 75

Studies of the biosynthesis of ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis V, have shown that the C-3 deoxygenation is a process consisting of two enzymatic steps. The first enzyme involved in this transformation is CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is a pyridoxamine 5'-phosphate dependent iron-sulfur protein. The second catalyst, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase, formally called CDP-6-deoxy-delta(3,4)-glucoseen reductase (E3), is an NADH dependent plant type [2Fe-2S] containing flavoenzyme. To better understand the electron transfer carried out by these two enzymes, the potentials of the E1 and E3 redox cofactors were determined spectroelectrochemically. At pH 7.5, the midpoint potential of the E3 FAD was found to be -212 mV, with the FADox/FADsq couple (E1o') and the FADsq/FADhq couple (E2o') calculated to be -231 and -192 mV, respectively. However, the E1o' and E2o' of the FAD in E3(apoFeS) at pH 7.5 were estimated to be -215 and -240 mV, respectively, which are quite different from those of the holo-E3, suggesting a significant effect of the iron-sulfur center on the redox properties of the flavin coenzyme. Our data also showed that the midpoint potential of the E3 iron-sulfur is -257 mV and that of the E1 [2Fe-2S] center is -209 mV. These values indicated a thermodynamic barrier to the proposed electron transfer of NADH->FAD=>E3[2Fe-2S]->E1[2Fe-2S] at pH 7.5. Regulation of electron transfer by several mechanisms is possible and experiments were performed to examine ways of overcoming the unfavorable electron transfer energetics in the E1/E3 system. It was found that both binding of E3 with NAD+ and complex formation between E3 and E1 showed no effect on the midpoint potentials of the E3 FAD and iron-sulfur center. Interestingly, the midpoint potential of the E3 FAD shifts dramatically to -273 mV (E1o' approximately -345 mV and E2o' approximately -200 mV) at pH 8.4, with very little semiquinone stabilization (< 5%). The potential of the E3 [2Fe-2S] center at pH 8.4 was also found to undergo a negative shift to -279 mV, and that of the E1 iron sulfur center remained essentially the same at -206 mV. These data indicated that the redox properties of this system may be regulated by pH and the electron transfer between the E3 redox centers may be prototropically controlled. These results also demonstrated that E3 is unique among this class of enzymes.
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PMID:Studies of the redox properties of CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1) and CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3): two important enzymes involved in the biosynthesis of ascarylose. 867 89


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