Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene.
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ABSTRACT:
SUBMITTER: Hyman MR
PROVIDER: S-EPMC1144898 | biostudies-other | 1985 May
REPOSITORIES: biostudies-other
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Project description:Photoinactivation of ammonia oxidation by Nitrosomonas europaea cells by near-u.v. light was confirmed and further shown to occur with the same rate constant as loss of bromoethane-oxidation activity. Hydroxylamine oxidation was much less photosensitive. Protection against inactivation was afforded by anaerobiosis, organic substrates of ammonia mono-oxygenase such as bromoethane, or metal-ion-chelating agents such as thiourea. The presence of 10 mM-NH4+ or 1 mM-hydroxylamine made little difference, whereas hydrazine had a potentiating effect. Illumination of cells also caused a bleaching in the absorption spectrum around 380 nm, along with changes in the cytochrome gamma-band region. Similar effects below 400 nm were obtained when organic substrates and inhibitors of the mono-oxygenase were added to cells in the dark. The copper proteins haemocyanin and tyrosinase have a photosensitive oxygenated state with a near-u.v. absorption band of similar half-width. They also have a sensitivity to chelating agents similar to that of ammonia mono-oxygenase. The experimental results are explained in terms of a three-stage catalytic cycle analogous to that for tyrosinase. In resting cells most of the enzyme is believed to be in an oxygenated (Oxy) form, which absorbs maximally at 378 nm and is photosensitive. In the presence of a substrate, one O atom is inserted into the substrate and the other is reduced to water, leaving the enzyme in an oxidized (Met) state. This is followed by a two-electron reduction of the proposed binuclear copper site to give a reduced (Deoxy) state, which can bind O2 to complete the cycle.
| S-EPMC1144737 | biostudies-other
Project description:pMMO (particulate methane mono-oxygenase) is an integral membrane metalloenzyme that catalyses the oxidation of methane to methanol. The pMMO metal active site has not been identified, precluding detailed investigation of the reaction mechanism. Models for the metal centres proposed by various research groups have evolved as crystallographic and spectroscopic data have become available. The present review traces the evolution of these active-site models before and after the 2005 Methylococcus capsulatus (Bath) pMMO crystal structure determination.
| S-EPMC3362633 | biostudies-literature
Project description:Changes in metabolite levels of the kynurenine pathway have been observed in patients with CKD, suggesting involvement of this pathway in disease pathogenesis. Our recent genetic analysis in the mouse identified the kynurenine 3-mono-oxygenase (KMO) gene (Kmo) as a candidate gene associated with albuminuria. This study investigated this association in more detail. We compared KMO abundance in the glomeruli of mice and humans under normal and diabetic conditions, observing a decrease in glomerular KMO expression with diabetes. Knockdown of kmo expression in zebrafish and genetic deletion of Kmo in mice each led to a proteinuria phenotype. We observed pronounced podocyte foot process effacement on long stretches of the filtration barrier in the zebrafish knockdown model and mild podocyte foot process effacement in the mouse model, whereas all other structures within the kidney remained unremarkable. These data establish the candidacy of KMO as a causal factor for changes in the kidney leading to proteinuria and indicate a functional role for KMO and metabolites of the tryptophan pathway in podocytes.
| S-EPMC5084883 | biostudies-other
Project description:Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.
| S-EPMC3304488 | biostudies-literature
Project description:YdhR is a 101-residue conserved protein from Escherichia coli. Sequence searches reveal that the protein has >50% identity to proteins found in a variety of other bacterial genomes. Using size exclusion chromatography and fluorescence spectroscopy, we determined that ydhR exists in a dimeric state with a dissociation constant of approximately 40 nM. The three-dimensional structure of dimeric ydhR was determined using NMR spectroscopy. A total of 3400 unambiguous NOEs, both manually and automatically assigned, were used for the structure calculation that was refined using an explicit hydration shell. A family of 20 structures was obtained with a backbone RMSD of 0.48 A for elements of secondary structure. The structure reveals a dimeric alpha,beta fold characteristic of the alpha+beta barrel superfamily of proteins. Bioinformatic approaches were used to show that ydhR likely belongs to a recently identified group of mono-oxygenase proteins that includes ActVA-Orf6 and YgiN and are involved in the oxygenation of polyaromatic ring compounds.
| S-EPMC2253234 | biostudies-literature
Project description:AimThe aim of this study was to investigate intra- and inter-individual variations of functional metabolic capacity of flavin-containing mono-oxygenase (FMO) during childhood using trimethylamine N-oxygenation as a probe reaction.MethodsTrimethylamine N-oxygenation functional activity and presence of FMO1 (fetal form), FMO3 (adult form), and FMO5 (endogenous form) were immunochemically determined and compared in human liver microsomes obtained from children at various ages. As a control, the same parameters were studied with recombinant FMO1, FMO3 and FMO5 proteins as enzyme sources. Developmental variation in functional metabolic capacity of FMO was estimated by measuring urinary trimethylamine and its N-oxide in several individuals at different ages and in a group of 77 subjects in childhood.ResultsThere was a significant correlation between trimethylamine N-oxygenation functional activity and FMO3 expression levels in human liver microsomes (r= 0.71, P < 0.05, n= 9). Trimethylamine N-oxygenation was catalyzed largely by FMO3 and not by FMO1 or FMO5. On the basis of analysis of intra-individual observations and collective urine samples under daily dietary conditions it was possible that neonates or infants harbouring at least one non-inactive-allele of the FMO3 gene could have developmental FMO3 metabolic capacity in childhood.ConclusionsDevelopmental variations in functional metabolic capacity of FMO3 in childhood were shown both on the basis of in vivo phenotyping tests and in in vitro liver microsomal determinations.
| S-EPMC3080647 | biostudies-literature
Project description:Osteoarthritis (OA) is a chronic progressive osteoarthropathy in the elderly. Osteoclast activation plays a crucial role in the occurrence of subchondral bone loss in early OA. However, the specific mechanism of osteoclast differentiation in OA remains unclear. In our study, gene expression profiles related to OA disease progression and osteoclast activation were screened from the Gene Expression Omnibus (GEO) repository. GEO2R and Funrich analysis tools were employed to find differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that chemical carcinogenesis, reactive oxygen species (ROS), and response to oxidative stress were mainly involved in osteoclast differentiation in OA subchondral bone. Furthermore, fourteen DEGs that are associated with oxidative stress were identified. The first ranked differential gene, heme oxygenase 1 (HMOX1), was selected for further validation. Related results showed that osteoclast activation in the pathogenesis of OA subchondral bone is accompanied by the downregulation of HMOX1. Carnosol was revealed to inhibit osteoclastogenesis by targeting HMOX1 and upregulating the expression of antioxidant protein in vitro. Meanwhile, carnosol was found to alleviate the severity of OA by inhibiting the activation of subchondral osteoclasts in vivo. Our research indicated that the activation of osteoclasts due to subchondral bone redox dysplasia may serve as a significant pathway for the advancement of OA. Targeting HMOX1 in subchondral osteoclasts may offer novel insights for the treatment of early OA.
| S-EPMC11199094 | biostudies-literature
Project description:Regio- and stereoselective oxidation of an unactivated C-H bond remains a central challenge in organic chemistry. Considerable effort has been devoted to identifying transition metal complexes, biological catalysts, or simplified mimics, but limited success has been achieved. Cytochrome P450 mono-oxygenases are involved in diverse types of regio- and stereoselective oxidations, and represent a promising biocatalyst to address this challenge. The application of this class of enzymes is particularly significant if their substrate spectra can be broadened, selectivity controlled, and reactions catalyzed in the absence of expensive heterologous redox partners. In this study, we engineered a macrolide biosynthetic P450 mono-oxygenase PikC (PikC(D50N)-RhFRED) with remarkable substrate flexibility, significantly increased activity compared to wild-type enzyme, and self-sufficiency. By harnessing its unique desosamine-anchoring functionality via a heretofore under-explored "substrate engineering" strategy, we demonstrated the ability of PikC to hydroxylate a series of carbocyclic rings linked to the desosamine glycoside via an acetal linkage (referred to as "carbolides") in a regioselective manner. Complementary analysis of a number of high-resolution enzyme-substrate cocrystal structures provided significant insights into the function of the aminosugar-derived anchoring group for control of reaction site selectivity. Moreover, unexpected biological activity of a select number of these carbolide systems revealed their potential as a previously unrecorded class of antibiotics.
| S-EPMC2774028 | biostudies-literature
Project description:Many bioactive polypeptides or neuropeptides possess a C-terminal alpha-amide group as a critical determinant for their optimal bioactivities. The amide functions are introduced by the sequential actions of peptidylglycine alpha-hydroxylating mono-oxygenase (PHM; EC 1.14.17.3) and peptidylamidoglycollate lyase (PAL; EC 4.3.2.5) from their glycine-extended precursors. In the present study we examined the kinetic isotope effects of the frog PHM reaction by competitive and non-competitive approaches. In the competitive approach we employed the double-label tracer method with D-Tyr-[U-14C]Val-Gly, D-Tyr-[3,4-3H]Val-[2,2-2H2]-Gly, and D-Tyr-Val-(R,S)[2-3H]Gly as substrates, and we determined the deuterium and tritium effects on Vmax/Km as 1.625+/-0.041 (mean+/-S. D.) and 2.71+/-0.16 (mean+/-S.D.), respectively. The intrinsic deuterium isotope effect (Dk) on the glycine hydroxylation reaction was estimated to be 6.5-10.0 (mean 8.1) by the method of Northrop [Northrop (1975) Biochemistry 14, 2644-2651]. In the non-competitive approach with N,N-dimethyl-1,4-phenylenediamine as a reductant, however, the deuterium effect on Vmax (DV) was approximately unity, although the deuterium effect on Vmax/Km (DV/K) was comparable to that obtained by the competitive approach. These results indicated that DV was completely masked by the presence of one or more steps much slower than the glycine hydroxylation step and that DV/K was diminished from Dk by a large forward commitment to catalysis. The addition of PAL, however, increased the apparent DV from 1.0 to 1.2, implying that the product release step was greatly accelerated by PAL. These results suggest that the product release is rate-limiting in the overall PHM reaction. The large Dk indicated that the glycine hydroxylation catalysed by PHM might proceed in a stepwise mechanism similar to that proposed for the dopamine beta-hydroxylase reaction [Miller and Klinman (1983) Biochemistry 22, 3091-3096].
| S-EPMC1219851 | biostudies-other
Project description:The enzyme catalysing the hydroxylation of ecdysone to 20-hydroxyecdysone, ecdysone 20-mono-oxygenase (EC 1.14.99.22), was investigated in the Malpighian tubules of fifth-instar locusts, Schistocerca gregaria. Enzyme activity was optimal at 35 degrees C and pH 6.8-8.0. Under these conditions the mono-oxygenase exhibited an apparent Km for ecdysone of 7.1 X 10(-7) M, a maximal specific activity of 1.1 nmol/h per mg of protein and was competitively inhibited by 20-hydroxyecdysone with an apparent Ki of 6.3 X 10(-7) M. Enzyme activity was decreased in the presence of Ca2+, Mg2+, EDTA and non-ionic detergents. The Malpighian tubule ecdysone 20-mono-oxygenase was localized primarily in the subcellular fraction sedimenting at 7500 g and, on the basis of marker enzyme profiles, was assigned mainly to the mitochondria. NADPH was required for activity, although addition of NADH together with NADPH had a synergistic effect. NADP+-dependent isocitrate dehydrogenase (EC 1.1.1.42) and an energy-dependent NAD(P) transhydrogenase (EC 1.6.1.1.) appeared to be the major sources of reducing equivalents, with the contribution from the 'malic enzyme' (EC 1.1.1.40) being less important. The monooxygenase was characterized as a cytochrome P-450-containing mixed-function oxidase from the inhibition patterns with metyrapone, CO and cyanide; CO inhibition was reversible with monochromatic light at 450 nm. However, the ecdysone 20-mono-oxygenase shows much lower sensitivity to CO inhibition and to photodissociation of the CO-inhibited complex than do vertebrate cytochrome P-450-dependent hydroxylation systems. The concentration of cytochrome P-450 in the Malpighian tubule mitochondria was 30 pmol/mg of protein. The properties of the mono-oxygenase are discussed in relation to hydroxylation enzymes from other sources.
| S-EPMC1144370 | biostudies-other