Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two separate enzymes, which determine resistance to inorganic mercury and organomercurials, have been purified from the plasmid-bearing Escherichia coli strain J53-1(R831). The mercuric reductase that reduces Hg2+ to volatile Hg0 was purified about 240-fold from the 160,000 X g supernatant of French press disrupted cells. This enzyme contains bound FAD, requires NADPH as an electron donor, and requires the presence of a sulfhydryl compound for activity. The reductase has a Km of 13 micron HgCl2, a pH optimum of 7.5 in 50 mM sodium phosphate buffer, an isoelectric point of 5.3, a Stokes radius of 50 A, and a molecular weight of about 180,000. The subunit molecular weight, determined by gel electrophoresis in the presence of sodium dodecyl sulfate, is about 63,000 +/- 2,000. These results suggest that the native enzyme is composed of three identical subunits. The organomercurial hydrolase, which breaks the mercury-carbon bond in compounds such as methylmercuric chloride, phenylmercuric acetate, and ethylmercuric chloride, was purified about 38-fold over the starting material. This enzyme has a Km of 0.56 micron for ethylmercuric chloride, a Km of 7.7 micron for methylmercuric chloride, and two Km values of 0.24 micron and over 200 micron for phenylmercuric acetate. The hydrolase has an isoelectric point of 5.5, requires the presence of EDTA and a sulfhydryl compound for activity, has a Stokes radius of 24 A, and has a molecular weight of about 43,000 +/- 4,000.
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PMID:The mercuric and organomercurial detoxifying enzymes from a plasmid-bearing strain of Escherichia coli. 35 Aug 72

A ketone monooxygenase was purified from cells of Pseudomonas cepacia grown on 2-tridecanone as sole carbon source. Enzyme stability is maintained by the addition of ethanol, EDTA, and dithiothreitol. Stoichiometric studies show that for 1 mol of undecyl acetate formed, 1 mol of O2 is consumed and 1 mol of NADPH is oxidized. The monooxygenase, purified to homogeneity, has a molecular weight of approximately 123,000 and consists of two equal subunits with molecular weights of 55,000. The enzyme contains FAD and exhibits absorption maxima at 375 and 488 nm. Enzyme activity is inhibited by thiol-active reagents and the inhibition by the cations, cadmium, copper, zinc, and mercury, is reversed by dithiothreitol, indicating the presence of essential sulfhydryl groups. Substrate specificity tests show that acetate esters are formed from methyl ketones from C-7 through C-14. The oxygenase is also active on isomers of 2-tridecanone forming esters from 3- through 7-tridecanone. With 6-tridecanone, two esters are formed, heptyl hexanoate and pentyl octanoate, indicating that oxygen is inserted on either side of the carbonyl group. In addition, the enzyme catalyzes the lactonization of the cyclic ketone, cyclopentanone, with the formation of 5-valerolactone.
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PMID:A novel ketone monooxygenase from Pseudomonas cepacia. Purification and properties. 92 12

Environmental and clinical isolates of mercury-resistant (resistant to inorganic mercury salts and organomercurials) bacteria have genes for the enzymes mercuric ion reductase and organomercurial lyase. These genes are often plasmid-encoded, although chromosomally encoded resistance determinants have been occasionally identified. Organomercurial lyase cleaves the C-Hg bond and releases Hg(II) in addition to the appropriate organic compound. Mercuric reductase reduces Hg(II) to Hg(O), which is nontoxic and volatilizes from the medium. Mercuric reductase is a FAD-containing oxidoreductase and requires NAD(P)H and thiol for in vitro activity. The crystal structure of mercuric ion reductase has been partially solved. The primary sequence and the three-dimensional structure of the mercuric reductase are significantly homologous to those of other flavin-containing oxidoreductases, e.g., glutathione reductase and lipoamide dehydrogenase. The active site sequences are the most conserved region among these flavin-containing enzymes. Genes encoding other functions have been identified on all mercury ion resistance determinants studied thus far. All mercury resistance genes are clustered into an operon. Hg(II) is transported into the cell by the products of one to three genes encoded on the resistance determinants. The expression of the operon is regulated and is inducible by Hg(II). In some systems, the operon is inducible by both Hg(II) and some organomercurials. In gram-negative bacteria, two regulatory genes (merR and merD) were identified. The (merR) regulatory gene is transcribed divergently from the other genes in gram-negative bacteria. The product of merR represses operon expression in the absence of the inducers and activates transcription in the presence of the inducers. The product of merD coregulates (modulates) the expression of the operon. Both merR and merD gene products bind to the same operator DNA. The primary sequence of the promoter for the polycistronic mer operon is not ideal for efficient transcription by the RNA polymerase. The -10 and -35 sequences are separated by 19 (gram-negative systems) or 20 (gram-positive systems) nucleotides, 2 or 3 nucleotides longer than the 17-nucleotide optimum distance for binding and efficient transcription by the Escherichia coli sigma 70-containing RNA polymerase. The binding site of MerR is not altered by the presence of Hg(II) (inducer). Experimental data suggest that the MerR-Hg(II) complex alters the local structure of the promoter region, facilitating initiation of transcription of the mer operon by the RNA polymerase. In gram-positive bacteria MerR also positively regulates expression of the mer operon in the presence of Hg(II).
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PMID:Bacterial resistances to inorganic mercury salts and organomercurials. 131 Nov 13

Mercuric ion reductase (MerA) catalyzes the reduction of Hg(II) to Hg(0) as the last step in the bacterial mercury detoxification pathway. A member of the flavin disulfide oxidoreductase family, MerA contains an FAD prosthetic group and redox-active disulfide in its active site. However, the presence of these two moieties is not sufficient for catalytic Hg(II) reduction, as other enzyme family members are potently inhibited by mercurials. We have previously identified a second pair of active site cysteines (Cys558 Cys559 in the Tn501 enzyme) unique to MerA, that are essential for high levels of mercuric ion reductase activity [Moore, M. J., & Walsh, C. T. (1989) Biochemistry 28, 1183; Miller, S. M., et al. (1989) Biochemistry 28, 1194]. In this paper, we have examined the individual roles of Cys558 and Cys559 by site-directed mutagenesis of each to alanine. Phenotypic analysis indicates that both merA mutations result in a total disruption of the Hg(II) detoxification pathway in vivo, while characterization of the purified mutant enzymes in vitro shows each to have differential effects on catalytic function. Compared to wild-type enzyme, the C558A mutant shows a 20-fold reduction in kcat and a 10-fold increase in Km, for an overall decrease in catalytic efficiency of 200-fold in kcat/Km. In contrast, mutation of Cys559 to alanine results in less than a 2-fold reduction in kcat and an increase in Km of only 4-5 fold for an overall decrease in catalytic efficiency of only ca. 10-fold in vitro. From these results, it appears that Cys558 plays a more important role in forming the reducible complex with Hg(II), while both Cys558 and Cys559 seem to be involved in efficient scavenging (i.e., tight binding) of Hg(II).
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PMID:C-terminal cysteines of Tn501 mercuric ion reductase. 153 Dec 97

Several hundred million tons of toxic mercurials are dispersed in the biosphere. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase and mercuric ion reductase (MerA). The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases, catalyses the reaction NADPH + Hg(II)----NADP+ + H+ + Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), p1258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn501 and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon. These domains can be proteolytically cleaved off without changing the catalytic efficiency. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.
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PMID:Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607. 206 68

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be "linked" together on multiple resistance plasmids. For Cd2+, AsO2-, AsO4(3)-, Hg2+, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. The cadA ATPase of S. aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd2+ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K+ ATPases of Escherichia coli and Streptococcus faecalis, the H+ ATPases of yeast and Neurospora, the Na+/K+ ATPases of vertebrate animals, and the Ca2+ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in the mer system include a DNA-binding regulatory protein from the merR gene and a Hg2+ transport system consisting of a periplasmic Hg2(+)-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.
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PMID:DNA sequence analysis of bacterial toxic heavy metal resistances. 248 81

The mercurial-resistance determinant from Staphylococcus aureus plasmid pI258 is located on a 6.4-kilobase-pair Bgl II fragment. The determinant was cloned into both Bacillus subtilis and Escherichia coli. Mercury resistance was found only in B. subtilis. The 6404-base-pair DNA sequence of the Bgl II fragment was determined. The mer DNA sequence includes seven open reading frames, two of which have been identified by homology with the merA (mercuric reductase) and merB (organomercurial lyase) genes from the mercurial-resistance determinants of Gram-negative bacteria. Whereas 40% of the amino acid residues overall were identical between the pI258 merA polypeptide product and mercuric reductases from Gram-negative bacteria, the percentage identity in the active-site positions and those thought to be involved in NADPH and FAD contacts was above 90%. The 216 amino acid organomercurial lyase sequence was 39% identical with that from a Serratia plasmid, with higher conservation in the middle of the sequences and lower homologies at the amino and carboxyl termini. The remaining five open reading frames in the pI258 mer sequence have no significant homologies with the genes from previously sequenced Gram-negative mer operons.
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PMID:Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258. 303 34

Bacteria mediate resistance to organomercurial and inorganic mercuric salts by metabolic conversion to nontoxic elemental mercury, Hg(0). The genes responsible for mercury resistance are organized in the mer operon, and such operons are often found in plasmids that also bear drug resistance determinants. We have subcloned three of these mer genes, merR, merB, and merA, and have studied their protein products via protein overproduction and purification, and structural and functional characterization. MeR is a metalloregulatory DNA-binding protein that acts as a repressor of both its own and structural gene transcription in the absence of Hg(II); in addition it acts as a positive effector of structural gene transcription when Hg(II) is present. MerB, organomercury lyase, catalyzes the protonolytic fragmentation of organomercurials to the parent hydrocarbon and Hg(II) by an apparent SE2 mechanism. MerA, mercuric ion reductase, is an FAD-containing and redox-active disulfide-containing enzyme with homology to glutathione reductase. It has evolved the unique catalytic capacity to reduce Hg(II) to Hg(0) and thereby complete the detoxification scheme.
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PMID:Molecular basis of bacterial resistance to organomercurial and inorganic mercuric salts. 327 86

The flavoprotein mercuric reductase catalyzes the two-electron reduction of mercuric ions to elemental mercury using NADPH as an electron donor. It has now been purified from Pseudomonas aeruginosa PAO9501 carrying the plasmid pVS1. In this plasmid system, where the mer operon is on the transposon Tn501, mercuric reductase comprises up to 6% of the soluble cellular protein upon induction with mercurials. The purification is a rapid (two-step), high yield (80%) procedure. Anaerobic titrations of mercuric reductase with dithionite revealed the formation of a charge transfer complex with an absorbance maximum around 540 nm. Striking spectroscopic similarities to lipoamide dehydrogenase and glutathione reductase were observed. These two enzymes, which catalyze the transfer of electrons between pyridine nucleotides and disulfides, are flavoproteins which contain an oxidation-reduction-active cysteine residue at the active site. The expectation that mercuric reductase contains a similar electron acceptor was confirmed when it was shown that mercuric reductase has the capacity to accept four electrons per FAD-containing subunit, and that two thiols become kinetically titrable by 5,5'-dithiobis-(2-nitrobenzoate) upon reduction with NADPH. These are characteristic features of the disulfide reductase class of flavoproteins. Further similarities with at least one of these enzymes, lipoamide dehydrogenase, include the E/EH2 midpoint potential (-269 mV), fluorescence properties, and extinction coefficients of E and EH2. Preliminary observations relevant to an understanding of the mechanism of mercuric reductase are discussed.
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PMID:Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide. 627

2-Methyl-branched chain acyl-CoA dehydrogenase was purified to homogeneity from rat liver mitochondria. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both with and without 2-mercaptoethanol, the enzyme showed a single protein band with Mr = 41,500, suggesting that this enzyme is composed of four subunits of equal size. Its isoelectric point was 5.50 +/- 0.2, and A1%280 nm was 12.5. This enzyme contained protein-bound FAD. The purified enzyme dehydrogenated S-2-methylbutyryl-CoA and isobutyryl-CoA with equal activity. The activities with each of these compounds were co-purified throughout the entire purification procedure. This enzyme also dehydrogenated R-2-methylbutyryl-CoA, but the specific activity was considerably lower (22%) than that for the S-enantiomer. The enzyme did not dehydrogenate other acyl-CoAs, including isovaleryl-CoA, propionyl-CoA, butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA, at any significant rate. Apparent Km and Vmax values for S-2-methylbutyryl-CoA were 20 microM and 2.2 mumol min-1 mg-1, respectively, while those for isobutyryl-CoA were 89 microM and 2.0 mumol min-1 mg-1 using phenazine methosulfate as an artificial electron acceptor. The enzyme was also active with electron transfer flavoprotein. Tiglyl-CoA and methacrylyl-CoA were identified as the reaction products from S-2-methylbutyryl-CoA and isobutyryl-CoA, respectively. 2-Ethylacrylyl-CoA was produced from R-2-methylbutyryl-CoA. Tiglyl-CoA competitively inhibited the activity with both S-2-methylbutyryl-CoA and isobutyryl-CoA with a similar Ki. The enzyme activity was also severely inhibited by several organic sulfhydryl reagents such as N-ethylmaleimide, p-hydroxymercuribenzoate, and methyl mercury iodide. The pattern and degree of inhibition were essentially identical for both substrates. The purified 2-methyl-branched chain acyl-CoA dehydrogenase was immunologically distinct from isovaleryl-CoA-, short chain acyl-CoA-, medium chain acyl-CoA-, or long chain acyl-CoA dehydrogenase.
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PMID:Purification and characterization of 2-methyl-branched chain acyl coenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria. 687 97


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