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PubMed2001

Supramolecular assembly and acid resistance of Helicobacter pylori urease.

Helicobacter pylori, an etiologic agent in a variety of gastroduodenal diseases, produces a large amount of urease, which is believed to neutralize gastric acid by producing ammonia for the survival of the bacteria. Up to 30% of the enzyme associates with the surface of intact cells upon lysis of neighboring bacteria. The role of the enzyme at the extracellular location has been a subject of controversy because the purified enzyme is irreversibly inactivated below pH 5. We have determined the crystal structure of H. pylori urease, which has a 1.1 MDa spherical assembly of 12 catalytic units with an outer diameter of approximately 160 A. Under physiologically relevant conditions, the activity of the enzyme remains unaffected down to pH 3. Activity assays under different conditions indicated that the cluster of the 12 active sites on the supramolecular assembly may be critical for the survival of the enzyme at low pH. The structure provides a novel example of a molecular assembly adapted for acid resistance that, together with the low Km value of the enzyme, is likely to enable the organism to inhabit the hostile niche.

Amino Acid Sequence↗

PubMed2000

Inhibition by unsaturated fatty acids of type II secretory phospholipase A2 synthesis in guinea-pig alveolar macrophages evidence for the eicosanoid-independent pathway.

The effect of arachidonic acid (C20:4) on the production of secretory type II phospholipase A2 (sPLA2-II) by guinea-pig alveolar macrophages was investigated. We show that incubation of these cells with 1-30 microM of arachidonic acid inhibits the synthesis of sPLA2-II in a concentration-dependent manner with an IC50 of approximately 7.5 microM. The inhibition by low concentrations (5 microM) of arachidonic acid was partially reduced by pretreatment of alveolar macrophages with cyclooxygenase or cytochrome P450 inhibitors (aspirin and 1-aminobenzotriazole, respectively), but not by lipoxygenase inhibitor, BW A4C. However, these inhibitors failed to interfere with the effect of high concentrations (30 microM) of arachidonic acid, suggesting that the latter may act on the expression of sPLA2-II, at least in part, independently of eicosanoid generation. Indeed, a similar inhibitory effect on sPLA2-II activity and mRNA expression was observed with other unsaturated fatty acids such as eicosapentaenoic (C20:5) and oleic (C18:1) acids, but not with the saturated fatty acid, palmitic acid (C16:0). In addition, arachidonic acid partially reduced the secretion of tumor necrosis factor alpha, an important intermediate in the induction of sPLA2-II synthesis by guinea-pig alveolar macrophages. However, addition of recombinant tumor necrosis factor alpha failed to reverse the inhibitory effect of arachidonic acid on sPLA2-II expression, suggesting that this process occurs downstream of tumor necrosis factor alpha secretion. We conclude that the expression of sPLA2-II in alveolar macrophages is down-regulated at the transcriptional level by arachidonic acid either directly or via its cyclooxygenase and cytochrome P450-derived metabolites.

Animals↗

PubMed2002

Cloning and characterization of a leucyl aminopeptidase from three pathogenic Leishmania species.

Aminopeptidases are emerging as exciting novel drug targets and vaccine candidates in parasitic infections. In this study, we describe for the first time an aminopeptidase from three highly pathogenic Leishmania species. Intronless genes encoding a leucyl aminopeptidase (lap) were cloned from Leishmania amazonensis, Leishmania donovani, and Leishmania major, which encoded 60-kDa proteins that displayed homology to leucyl aminopeptidases from Gram-negative bacteria, plants, and mammals. The lap genes were present as a single copy in each genome, and lap mRNA was detected by reverse transcription-PCR in all life-cycle stages of L. amazonensis. Lap assembled into catalytically competent 360-kDa hexamers and demonstrated potent amidolytic activity against synthetic aminopeptidase substrates containing leucine, methionine, and cysteine residues, representing the most restricted substrate specificity of any leucyl aminopeptidase described to date. Optimal activity was observed against L-leucyl-7-amido-4-methylcoumarin (k(cat)/K(m) approximately 63 s(-1) x mm(-1)) with a pH optimum of 8.5. Leishmania Lap activity was inhibited by metal ion chelators and enhanced by divalent manganese, cobalt, and nickel cations, although only zinc was detected in the purified Lap by inductively coupled plasma atomic emission spectroscopy, indicating that zinc is the natural Lap cofactor. Activity was potently inhibited by bestatin and apstatin in a slow binding competitive fashion, with K(i)* values of 3 and 44 nm, respectively. Actinonin was a tight binding competitive inhibitor (K(i) approximately 1 nm), whereas arphamenine A (K(i) approximately 70 microm) and L-leucinol (K(i) approximately 100 microm) were non-tight binding competitive inhibitors. Lap was not secreted by Leishmania in vitro and was localized to the parasite cytosol.

Amino Acid Sequence↗

PubMed1986

Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli.

A cloned 8.3-kilobase-pair DNA fragment carrying all the genes (iucABCD iutA) of the aerobactin iron transport system of plasmid pColV-K30 was subjected to in vitro mutagenesis to afford mutant genes iucA, iucC, and iucA iucC. Complementation analyses and identification of aerobactin precursors accumulated by Escherichia coli cells harboring the different constructions allowed assignment of the iucA and iucC genes to discrete steps in biosynthesis of the siderophore from N epsilon-acetyl-N epsilon-hydroxylysine and citrate. Plasmid pVLN10, a derivative carrying a DNA fragment complementing an iucC mutation, expressed in a minicell system a single 62,000-dalton protein as the product of this gene.

Bacteriocin Plasmids↗

PubMed1975

Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.

Alanine↗

PubMed1970