Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting k cat / K m on the order of 104 M-1 s-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2 ′ -phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.

Identification of the NADH-oxidase gene in Trichomonas vaginalis

The microaerophilic human parasite Trichomonas vaginalis causes infections in the urogenital tract and is one of the most often sexually transmitted pathogens worldwide. Due to its anaerobic metabolism, it has to quickly remove intracellular oxygen in order to avoid deactivation of essential metabolic enzymes such as oxygen-sensitive pyruvate:ferredoxin oxidoreductase (PFOR). Two major enzyme activities which are responsible for the removal, i.e. reduction, of molecular oxygen have been identified in T. vaginalis flavin reductase, formerly designated NADPH oxidase, which indirectly reduces oxygen to hydrogen peroxide via flavin mononucleotide (FMN), and NADH oxidase which reduces oxygen to water. Flavin reductase has been identified and characterized at the gene level as well as enzymatically, but NADH oxidase has so far only been characterized enzymatically with enzyme isolated from T. vaginalis cell extracts. In this study, we identified NADH oxidase by mass spectrometry after isolation of the enzyme from gel bands positively staining for NADH oxidase activity. In strain C1 (ATCC 30001) which is known to lack NADH oxidase activity completely, the NADH oxidase gene has a deletion at position 1540 of the open reading frame leading to a frame shift and, as a consequence, to premature termination of the encoded polypeptide.

Identification of bioactive compounds from Fraxinus angustifolia extracts with anti-NADH oxidase activity of bovine milk xanthine oxidoreductase

Fraxinus angustifolia leaves and bark are used in traditional medicine against various inflammatory-related pathologies incumbent to reactive oxygen species (ROS) generation by the NADH oxidase activity of enzymes such as xanthine oxidoreductase (XOR). This study was designed to investigate the in vitro and in vivo inhibitory activities of this enzyme by Fraxinus angustifolia extracts. The leaf organic phase of ethyl acetate (LFA) and its bark aqueous counterpart (BFA) showed the strongest anti-NADH oxidase activity in vitro (IC50 = 38.51 and 42.04 μg mL-1, respectively). They consequently suppressed superoxide generation both enzymatically (53% and 19%, respectively) and nonenzymatically (34% and 19%, respectively). These results were corroborated in vivo, with high anti- NADH oxidase potential of the leaves and bark extracts (75.32% and 51.32%, respectively) concomitant with moderate hypouricemic activities (36.84% and 38.59%, respectively). Bio-guided fractionation led to the identification, by LC-DAD-MS/MS, of esculin and calcelarioside in bark and kaempferol glucoside in leaves as the main compounds responsible for the anti-NADH oxidase activity of XOR. These results plead in favor of the use of F. angustifolia as a source of potentially interesting therapeutic substances.

Blockade of endogenous tissue kallikrein aggravates renal injury by enhancing oxidative stress and inhibiting matrix degradation

Levels of tissue kallikrein (TK) are significantly lower in the urine of patients with kidney failure, and TK expression is specifically diminished in rat kidney after recovery from ischemia-reperfusion injury. In this study, we investigated the functional consequence of blocking endogenous TK activity in a rat model of chronic kidney disease. Inhibition of endogenous TK levels for 10 days by neutralizing TK antibody injection in DOCA-salt rats caused a significant increase in blood urea nitrogen and urinary protein levels, and a decrease in creatinine clearance. Kidney sections from anti-TK antibody-treated rats displayed a marked rise in tubular dilation and protein cast accumulation as well as glomerular sclerosis and size. TK blockade also increased inflammatory cell infiltration, myofibroblast and collagen accumulation, and collagen fraction volume. Elevated renal inflammation and fibrosis by anti-TK antibody were associated with increased expression of tumor necrosis factor-α, intercellular adhesion molecule-1, tissue inhibitor of metalloproteinase-2 (TIMP-2), and plasminogen activator inhibitor-1 (PAI-1). Moreover, the detrimental effect of TK blockade resulted in reduced nitric oxide (NO) levels as well as increased serum lipid peroxidation, renal NADH oxidase activity, and superoxide formation. In cultured proximal tubular cells, TK inhibited angiotensin II-induced superoxide production and NADH oxidase activity via NO formation. In addition, TK markedly increased matrix metalloproteinase-2 activity with a parallel reduction of TIMP-2 and PAI-1 synthesis. These findings indicate that endogenous TK has the propensity to preserve kidney structure and function in rats with chronic renal disease by inhibiting oxidative stress and activating matrix degradation pathways.