Reduction of<i>N</i>-Hydroxy-sulfonamides, Including<i>N</i>-Hydroxy-valdecoxib, by the Molybdenum-Containing Enzyme mARC

Havemeyer, Antje; Grünewald, Sanja; Wahl, Bettina; Bittner, Florian; Mendel, Ralf R.; Erdélyi, Péter; Fischer, János; Clement, Bernd

doi:10.1124/dmd.110.032813

Cited by 45 publications

(44 citation statements)

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“…With the exception of the human Moco sulfurase HMCS, which is required for activating the Mo-enzymes AO and XOR (9) and which is the name-giving member of the MOSC family, no other homolog of hmARC-1 and hmARC-2 is found in the human genome. Cloning of hmARC-1 and hmARC-2 cDNAs derived from HepG2 cells (16,17) basically confirmed the predicted sequences of the protein-encoding open reading frames as deposited in the database, with the exception of two reproducible polymorphisms that were identified in the hmARC-1 cDNA causing substitutions of methionine 187 by lysine (protein entry NP_073583 versus AAH10619) and alanine 165 by threonine (protein entry ACB21046 versus NP_073583).…”

Section: Resultsmentioning

confidence: 93%

“…Construction of Expression Vectors-Full-length cDNAs of hmARC-1 (1011 base pairs corresponding to NCBI reference sequence NM_022746, MOSC-1) and hmARC-2 (1005 base pairs corresponding to NCBI reference sequence NM_017898, MOSC-2) were obtained as described earlier (15)(16)(17). For cloning of N-terminally truncated hmARC-1, the full-length open reading frame was truncated at its 5Ј end by PCR using the primers hmARC-1_Start_N-del (5Ј-ATA TAT GGA TCC ATG CAG CAG GTG GGC ACA GTG GCG-3Ј) and hmARC1_Stop (5Ј-AAA TTT AAG CTT TTA CTG GCC CAG CAG GTA CAC AGG-3Ј).…”

Section: Methodsmentioning

confidence: 99%

“…Determination of the N-Reductive Activity of hmARC-1 and hmARC-2-N-Reductive activities of hmARC-1 and hmARC-2 with benzamidoxime, N-hydroxy-L-arginine, or N-hydroxysulfonamides as substrates were determined as described previously with minor modifications (16,17). In the case of benzamidoxime, either 100 mM potassium phosphate buffer, pH 6.0 (for experiments shown in Figs.…”

Section: Expression and Purification Of Recombinant Proteins Used In mentioning

confidence: 99%

“…Thus, not only the cofactor composition but also the order of cofactors and the direction of electron flow are identical to NR. However, although many N-hydroxylated compounds have been found to serve as substrates for native and recombinant mARC proteins (15)(16)(17), the physiological substrates, and thus the physiological function, of mARC proteins are as yet unknown. The present study aimed to provide the first detailed biochemical information about the heterologously expressed human mARC isoforms hmARC-1 and hmARC-2 with particular focus on the characterization of their molybdenum center.…”

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Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

Wahl

Reichmann

Niks

et al. 2010

Journal of Biological Chemistry

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The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b 5 , and NADH/FADdependent cytochrome b 5 reductase. mARC proteins share a significant degree of homology to the molybdenum cofactorbinding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/ MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo-or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.In eukaryotes the trace element molybdenum is essential for a number of enzymes where the molybdenum atom is part of the so-called molybdenum cofactor (Moco) 2 in the active site of these enzymes (1). Moco is a pterin-based cofactor with a C6-substituted pyrano ring, a terminal phosphate, and a unique dithiolate group that binds the molybdenum atom. Moco-containing enzymes (Mo-enzymes) catalyze important reactions in the global carbon, sulfur, and nitrogen cycles that are characterized by transfer of an oxygen atom to or from a substrate. In mammals, one Mo-enzyme is sulfite oxidase (SO), which catalyzes the last step in the degradation of sulfur-containing amino acids and sulfatides (2). The active SO protein is a homodimer with each monomer of ϳ52 kDa consisting of a N-terminal cytochrome b 5 (cyt b 5 )/heme-binding domain and a C-terminal Moco-binding domain, the latter also harboring the dimerization interface. Both the Moco-and the heme-binding domain of mammalian SO are similar to the respective domains of nitrate reductase (NR), which catalyzes the first and rate-limiting step in nitrate assimilation in autotrophic organisms like plants, algae, and fungi (3). In addition to its N-terminal Mocobinding domain and the cytb 5 /heme-binding domain, each NR monomer possesses a C-terminal FAD-binding domain. Xanthine oxidoreductase (XOR) is another mammalian Mo-enzyme, and it is active as a homodimer with each ϳ145-kDa monomer consisting of several distinct domains: an N-terminal domain...

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Section: Resultsmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Expression and Purification Of Recombinant Proteins Used In mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

Wahl

Reichmann

Niks

et al. 2010

Journal of Biological Chemistry

Self Cite

101

155

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show abstract

“…The mechanism of this reduction has been extensively studied. Cytochrome b5 and its reductase and several molybdenumcontaining enzymes such as aldehyde oxidase were all capable of carrying out the reduction (Vonk et al, 1986;Saulter et al, 2005;Havemeyer et al, 2010). Sugihara et al (2000) also reported nonenzymatic N-dehydroxylation by hemoglobin under anaerobic conditions.…”

Section: Discussionmentioning

confidence: 99%

Investigation of Metabolism and Disposition of GSK1322322, a Peptidase Deformylase Inhibitor, in Healthy Humans Using the Entero-Test for Biliary Sampling

et al. 2014

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Aldehyde Reduction by Cytochrome P450

2011

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This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model α,β-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions.

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Reduction ofN-Hydroxy-sulfonamides, IncludingN-Hydroxy-valdecoxib, by the Molybdenum-Containing Enzyme mARC

Abstract: ABSTRACT:Purification of the mitochondrial enzyme responsible for reduction of N-hydroxylated amidine prodrugs led to the identification of two newly discovered mammalian molybdenum-containing proteins, the mitochondrial amidoxime reducing components mARC-1 and mARC-2 (

Cited by 45 publications

References 33 publications

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

Investigation of Metabolism and Disposition of GSK1322322, a Peptidase Deformylase Inhibitor, in Healthy Humans Using the Entero-Test for Biliary Sampling

Aldehyde Reduction by Cytochrome P450

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