Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690

An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O(2) as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp. KY 4690, a soil isolate, to an electrophoretically homogeneous state. The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa. The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family. The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5'-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa. Molecular oxygen served as the sole electron acceptor. These results suggest that aldehyde oxidase from Pseudomonas sp. KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum-molybdpterin-cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of [2Fe-2S] clusters per mol of enzyme protein. The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability.     

Mutational Analysis of the Multicopy hao Gene Coding for Hydroxylamine Oxidoreductase in Nitrosomonas sp. Strain ENI-11

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao ₁, hao ₂, and hao ₃) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao ₁::kan, hao ₂::kan, or hao ₃::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao ₁::kan and hao ₃::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.     

Light-induced changes in the amounts of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase and its mRNA in barley plants grown under a diurnal light/dark cycle

Seedlings of barley were grown either in continuous darkness or under a diurnal 12 h light/12 h dark cycle and the effects on NADPH-protochlorophyllide oxidoreductase were followed at two different levels. Firstly, the relative content of the mRNA encoding the NADPH-protochlorophyllide oxidoreductase was measured by dot-blot hybridization. Secondly, changes in the enzyme polypeptide were monitored either by the method of immunoblotting or by immunogold labelling of ultrathin sections of Lowicryl-embedded leaf tissue. Our results demonstrate that drastic diurnal changes in the level of mRNA sequences and the enzyme protein are unlikely to occur in plants which have been grown under natural light/dark conditions. In the dark, protein and mRNA accumulation occurs at an early developmental stage. These results are difficult to reconcile with the suggestion that the massive accumulation of mRNA and enzyme protein in dark-grown seedlings is primarily the consequence of an artificially extended darkperiod. In addition to the plastid-specific NADPH-protochlorophyllide oxidoreductase a closely related polypeptide has been detected outside the plastid in the surrounding cytoplasm (Dehseh et al. 1986b, Planta 169, 172–183). During the diurnal light/dark treatment of seedlings the concentrations of the two protein populations did not show any variation indicative of an exchange between the two protein populations across the plastid envelope.Abbreviation poly(A)⁺RNA polyadenylated RNA     

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process.     

Plastoquinones are effectively reduced by ferredoxin:NADP+ oxidoreductase in the presence of sodium cholate micelles. Significance for cyclic electron transport and chlororespiration

The effect of sodium cholate and other detergents (Triton X-100, sodium dodecyl sulphate, octyl glucoside, myristyltrimethylammonium bromide) on the reduction of plastoquinones (PQ) with a different length of the side-chain by spinach ferredoxin:NADP(+) oxidoreductase (FNR) in the presence of NADPH has been studied. Both NADPH oxidation and oxygen uptake due to plastosemiquinone autoxidation were highly stimulated only in the presence of sodium cholate among the used detergents. Sodium cholate at the concentration of 20 mM was found to be the most effective on both PQ-4 and PQ-9-mediated oxygen uptake. The FNR-dependent reduction of plastoquinones incorporated into sodium cholate micelles was stimulated by spinach ferredoxin but inhibited by Mg(2+) ions. It was concluded that the structure of sodium cholate micelles facilitates contact of plastoquinone molecules with the enzyme and creates favourable conditions for the reaction similar to those found in thylakoid membranes for PQ-9 reduction. The obtained results were discussed in terms of the function of FNR as a ferredoxin:plastoquinone reductase both in cyclic electron transport and chlororespiration.     

Kinetic model of disaccharide oxidation by Agrobacterium tumefaciens

Disaccharides were microbaially transformed to their corresponding 3-keto-derivatives by resting cells of Agrobacterium tumefaciens NCPPB 396. The kinetics and yield of this highly specific oxidation depend on several factors. The oxygen concentration especially has a major influence on the production of 3-keto-derivatives and was investigated kinetically with respect to low stationary oxygen concentrations in solution. Experiments showed unconventional results that conflicted with normal Michaelis-Menten kinetics. A kinetic model was developed and the kinetic constants were calculated. The model and experimental data for sucrose, maltose, iso-maltulose (palatinose), and leucrose are in good agreement with each other. Initial reaction rates with different sugars using constant oxygen concentrations resulted in a Michaelis-Mentent type function. The complete kinetics, including the effect of disaccharide and oxygen concentrations, are presented. (c) 1995 John Wiley & Sons, Inc.     

Catalysis by heteropentalenes of electron transfer to oxygen from ferredoxin-NADP oxidoreductase reduced by NADPH

A range of heteropentalene and bipyridinium compounds have been tested as catalysts of electron transfer to oxygen from spinach ferredoxin-NADP⁺ oxidoreductase reduced by NADPH. For a particular class of compound, the rate of oxygen reduction increased with increasing midpoint potential of the compound under conditions in which reduction of the compound was rate-limiting. Compounds with similar midpoint potentials from different structural classes showed marked differences in rate, attributed to specificity in the interaction with ferredoxin-NADP⁺ oxidoreductase.     

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180^T) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum. Received: 5 November 1997 / Accepted: 30 March 1998     

Mechanism of Inhibition of Xanthine Oxidoreductase by Allopurinol: Crystal Structure of Reduced Bovine Milk Xanthine Oxidoreductase Bound with Oxipurinol

Inhibitors of xanthine oxidoreductase block conversion of xanthine to uric acid and are therefore potentially useful for treatment of hyperuricemia or gout. We determined the crystal structure of reduced bovine milk xanthine oxidoreductase complexed with oxipurinol at 2.0 Å resolution. Clear electron density was observed between the N2 nitrogen of oxipurinol and the molybdenum atom of the molybdopterin cofactor, indicating that oxipurinol coordinated directly to molybdenum. Oxipurinol forms hydrogen bonds with glutamate802, arginine880, and glutamate1261, which have previously been shown to be essential for the enzyme reaction. We discuss possible differences in the hypouricemic effect of inhibitors, including allopurinol and newly developed inhibitors, based on their mode of binding in the crystal structures.