NADH and NADPH dehydrogenases from the blue-green alga,Aphanocapsa

Two different NAD(P)H dehydrogenases could be demonstrated in the blue-green alga, Aphanocapsa. Both function as quinone reductases using benzoquinone as electron acceptor. One, which was found in the soluble fraction, was NADH specific and showed high sensitivity to rotenone, thenoyltrifluoroacetone and o-phenanthroline. The second dehydrogenase was membrane-bound and used NADH as well as NADPH as substrates. Inhibition by rotenone and o-phenanthroline was less pronounced with the bound enzyme than with the soluble enzyme. Based on studies with NADH or NADPH, the membrane-bound enzyme apparently was associated with a low-temperature EPR signal at g=1.92 in the reduced state, indicative of an iron-sulfur center. The membrane-bound dehydrogenase was solubilized with Triton X-100 and partially purified. This preparation was used for studies of enzyme kinetics and acceptor specificity.Abbreviations DBMIB 2,5-dibromo methyl isopropylbenzoquinone - TTFA thenoyltrifluoroacetone - E _m midpoint redox potential     

Purine metabolites in serum of higher primates,including man

Using a new method for simultaneous quantification of hypoxanthine, xanthine, uric acid, and allantoin by means of high-pressure liquid chromatography, purine metabolites of 18 species of higher primates, including man, have been determined. The data thus produced indicate that the serum concentrations of purine metabolites in primates are influenced by nutrition, sexual hormones, and the procedures used in catching the animals for venipuncture. In the Callitrichidae examined, serum concentrations of purines differ significantly from species to species. The results of a nutritional test show that Callithrix jacchus possesses an efficient system for degradation of dietary purines.     

Characterization of a new marine nitrite oxidizing bacterium, Nitrospina watsonii sp. nov., a member of the newly proposed phylum “Nitrospinae”

Nitrite oxidizing bacteria are an integral part of the nitrogen cycle in marine waters, but the knowledge about their diversity is limited. Recently, a high abundance of Nitrospina-like 16S rRNA gene sequences has been detected in oceanic habitats with low oxygen content by molecular methods. Here, we describe a new strain of Nitrospina, which was sampled in 100 m depth from the Black Sea. It coexisted with a not-yet cultivated chemoorganotrophic gammaproteobacterium and could be purified by classical isolation methods including Percoll density gradient centrifugation. The new Nitrospina-like bacterium grew lithoautotrophically at 28 °C in diluted seawater supplemented with inorganic salts and nitrite. Gram-negative rods were characterized morphologically, physiologically and partly biochemically.     

Purine Composition of the Crystalline Cytoplasmic Inclusions of Paramecium tetraurelia

Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.     

Insights to the evolution of Nucleobase-Ascorbate Transporters (NAT/NCS2 family) from the Cys-scanning analysis of xanthine permease XanQ

The nucleobase-ascorbate transporter or nucleobase-cation symporter-2 (NAT/NCS2) family is one of the five known families of transporters that use nucleobases as their principal substrates and the only one that is evolutionarily conserved and widespread in all major taxa of organisms. The family is a typical paradigm of a group of related transporters for which conservation in sequence and overall structure correlates with high functional variations between homologs. Strikingly, the human homologs fail to recognize nucleobases or related cytotoxic compounds. This fact allows important biomedical perspectives for translation of structure-function knowledge on this family to the rational design of targeted antimicrobial purine-related drugs. To date, very few homologs have been characterized experimentally in detail and only two, the xanthine permease XanQ and the uric acid/xanthine permease UapA, have been studied extensively with site-directed mutagenesis. Recently, the high-resolution structure of a related homolog, the uracil permease UraA, has been solved for the first time with crystallography. In this review, I summarize current knowledge and emphasize how the systematic Cys-scanning mutagenesis of XanQ, in conjunction with existing biochemical and genetic evidence for UapA and the x-ray structure of UraA, allow insight on the structure-function and evolutionary relationships of this important group of transporters. The review is organized in three parts referring to (I) the theory of use of Cys-scanning approaches in the study of membrane transporter families, (II) the state of the art with experimental knowledge and current research on the NAT/NCS2 family, (III) the perspectives derived from the Cys-scanning analysis of XanQ.     

Screening and Inhibition Mechanism of Xanthine Oxidase Inhibitors in Ethanolic Extracts of Chimonanthus salicifolius Hu Leaves

This study aimed to evaluate the inhibition of the ethanol elutions of Chimonanthus salicifolius Hu leaves (CsHL) against xanthine oxidase (XO). The results of XO inhibition assay and enzymatic superoxide free radical scavenging assay in vitro showed that 70 % ethanol eluate (EE) had the best inhibitory effect and followed by 40 % EE. High performance liquid chromatograph analysis showed that quercetin and kaempferol were the potential active components of XO inhibition. The inhibition mechanism of quercetin and kaempferol on XO was investigated by kinetic analysis and fluorescence quenching titration assay. The molecular simulation further revealed that quercetin and kaempferol bind to XO mainly by hydrogen bonding and van der Waals, blocking the entry of substrates and leading to the inhibition of XO. In conclusion, the CsHL have inhibitory effects on XO activity, which provides a theoretical basis for relieving or preventing hyperuricemia and gout as a natural food or medicinal plant in the future.     

Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus

Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α₂β₂ hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched-chain 2-oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.     

Purification and characterization of formaldehyde dismutases of Methylobacterium sp. FD1

ABSTRACT

In the present study, we purified and characterized three formaldehyde dismutases (Fdms) (EC 1.2.98.1) (Fdm1, Fdm2, and Fdm3) of Methylobacterium sp. FD1. These Fdms (with His-tag) were produced in the recombinant E. coli and purified by immobilized metal affinity chromatography from the E. coli extracts. In each of the three Fdms, the enzyme-bound coenzyme was nicotinamide adenine dinucleotide (NAD(H)) and the enzyme-bound metal was zinc. The quaternary structures of these Fdms were estimated as homotetrameric. The optimal pHs and temperatures of Fdm1, Fdm2, and Fdm3 were approximately 6.5, 6.0, and 6.0, and 35°C, 25°C, and 30°C, respectively. The K_m values of Fdm1, Fdm2, and Fdm3 were 621, 865, and 414 mM, respectively. These results were similar to the properties of already-known Fdms. However, each of the Fdms of FD1 had methanol:p-nitroso-N,N-dimethylaniline oxidoreductase activity that is not found in already-known Fdms.     

Nonneuronal Localization for Steroid Converting Enzyme: 3α-Hydroxysteroid Oxidoreductase in Olfactory Tubercle of Rat Brain

3 alpha-Hydroxysteroid oxidoreductase (EC 1.1.1.50) was localized in the rat brain by cryostat sectioning, microassay, and neurochemical lesions. Single 16-microns sections were cut, homogenized, and assayed. In the olfactory tubercle 3 alpha-hydroxysteroid oxidoreductase activity is high in the piaglial layer at the surface, 20-fold lower at a depth of 50 microns, and 50-fold lower at a depth of 200 microns. A similar pattern of activity was seen in the olfactory bulb, the interpeduncular nucleus, the frontal pole of the cortex, and the frontoparietal cortex. When kainic acid, a toxin that destroys neurons but leaves glia and axons of passage intact, was injected into the olfactory tubercle, 3 alpha-hydroxysteroid oxidoreductase activity was undiminished whereas glutamic acid decarboxylase activity was reduced by 80%. This laminar distribution and insensitivity to kainic acid are consistent with a nonneuronal localization. The high concentration of astrocytes in the piaglial layer, where 3 alpha-hydroxysteroid oxidoreductase activity is highest, lead us to suggest that this enzyme is localized to astrocytes. The presence of particular enzymes in some brain regions and not in others determines which products are synthesized and which are inactivated in those regions. Thus, the location of 3 alpha-hydroxysteroid oxidoreductase and other steroid converting enzymes can affect the activity of neuronal circuits and the behaviours regulated by those circuits.     

A novel NADPH-dependent oxidoreductase with a unique domain structure in the hyperthermophilic Archaeon, Thermococcus litoralis

Thermococcus litoralis , a hyperthermophilic Archaeon, is able to reduce elemental sulfur during fermentative growth. An unusual gene cluster ( nsoABCD ) was identified in this organism. In silico analysis suggested that three of the genes ( nsoABC ) probably originated from Eubacteria and one gene ( nsoD ) from Archaea. The putative NsoA and NsoB are similar to NuoE- and NuoF-type electron transfer proteins, respectively. NsoC has a unique domain structure and contains a GltD domain, characteristic of glutamate synthase small subunits, which seems to be integrated into a NuoG-type sequence. Flavin and NAD(P)H binding sites and conserved cysteines forming iron–sulfur clusters binding motifs were identified in the protein sequences deduced. The purified recombinant NsoC contains one FAD cofactor per protein molecule and catalyzes the reduction of polysulfide with NADPH as an electron donor and it also reduces oxygen. It was concluded that the Nso complex is a new type of NADPH-oxidizing enzyme using sulfur and/or oxygen as an electron acceptor.