ATP phosphoribosyl transferase (ATP-PRT) joins ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP) in a highly regulated reaction that initiates histidine biosynthesis. The unusual hetero-octameric version of ATP-PRT includes four HisG S catalytic subunits based on the periplasmic binding protein fold and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. Here, we present the first structure of a PRPPbound ATP-PRT at 2.9 Å and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATPPRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface and new contacts between the HisZ motif 2 loop and the HisG S dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. Through the oligomerization and functional re-assignment of protein domains associated with aminoacylation and phosphate binding, the HisZ-HisG octameric ATP-PRT acquired the ability to initiate the synthesis of a key metabolic intermediate in an allosterically regulated fashion. Phosphoribosyl transferases (PRTs)5 catalyze the attack of a nitrogenous and/or aromatic base on 5-phosphoribosyl-1-pyrophosphate (PRPP), and thereby participate in essential reactions in the biosynthesis of nucleotides and the amino acids tryptophan and histidine (1, 2). The largest family (type I) of these structurally diverse enzymes includes many nucleotide salvage enzymes that share a five-stranded parallel  sheet fold with a substrate binding hood domain (3, 4). The folds of type II (5) and type III enzymes (6) are distinct from the class I enzymes, and the type III enzymes resemble nucleoside phosphorylase. Among the PRTs with complex quaternary structures and sophisticated regulation are the glutamine PRPP amidotransferase, which catalyzes the first committed step of purine biosynthesis (7), and ATP phosphoribosyl transferase (ATP-PRT), which joins ATP and PRPP to initiate synthesis of histidine (8, 9). Glutamine PRPP amidotransferase and ATP-PRT both exhibit pathway end product inhibition, and regulation by cellular energy levels (7, 10). ATP-PRT is competitively inhibited by AMP and ADP (8, 11-13) and non-competitively inhibited by histidine (Fig.
Many transition metal complexes mediate DNA oxidation in the presence of oxidizing radiation, photosensitizers, or oxidants. The final DNA oxidation products vary depending on the nature of metal complexes and the structure of DNA. Here we propose a mechanism of oxidation of a nucleotide, deoxyguanosine 5'-monophosphate (dGMP) by trans-d,l-1,2-diaminocyclohexanetetrachloroplatinum (trans-Pt(d,l)(1,2-(NH(2))(2)C(6)H(10))Cl(4), [Pt(IV)Cl(4)(dach)]; dach = diaminocyclohexane) to produce 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) stoichiometrically. The reaction was studied by high-performance liquid chromatography (HPLC), (1)H and (31)P nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). The proposed mechanism involves Pt(IV) binding to N7 of dGMP followed by cyclization via nucleophilic attack of a phosphate oxygen at C8 of dGMP. The next step is an inner-sphere, two-electron transfer to produce a cyclic phosphodiester intermediate, 8-hydroxyguanosine cyclic 5',8-(hydrogen phosphate). This intermediate slowly converts to 8-oxo-dGMP by reacting with solvent H(2)O.
The role of gastroesophageal reflux and micro-aspiration as a trigger of airways hyperresponsiveness (AHR) in patients with asthma is controversial. The role of acid reflux and aspiration as a direct cause of AHR in normal subjects is also unclear. We speculated that aspiration of a weak acid with a pH (1.8) equivalent to the upper range of typical gastric contents would lead to AHR in naive mice. We further speculated that modest reductions in aspirate acidity to a level expected during gastric acid suppression therapy (pH 4.0) would impede aspiration-induced AHR. BALB/c female mice were briefly anesthetized with isoflurane and allowed to aspirate 75 microl of saline with HCl (pH 1.8, 4.0, or 7.4) or underwent sham aspiration. Mice were re-anesthetized 2 or 24 h later, underwent tracheostomy, and were coupled to a mechanical ventilator. Forced oscillations were used to periodically measure respiratory impedance (Zrs) following aerosol delivery of saline and increasing doses of methacholine to measure for AHR. Values for elastance (H), airways resistance (R(N)), and tissue damping (G) were derived from Zrs. Aspirate pH of 1.8 led to a significant overall increase in peak R(N), G, and H compared with pH 4.0 and 7.4 at 2 and 24 h. Differences between pH 7.4 and 4.0 were not significant. In mice aspirating pH 1.8 compared with controls, airway lavage fluid contained more neutrophils, higher protein, and demonstrated higher permeability. We conclude that acid aspiration triggers an acute AHR, driven principally by breakdown of epithelial barrier integrity within the airways.
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