Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are flavoproteins which catalyze the oxidative demethylation of dimethylglycine to sarcosine and sarcosine to glycine, respectively. During these reactions tightly bound tetrahydropteroylpentaglutamate (H4PteGlu5) is converted to 5,10-methylene tetrahydropteroylpentaglutamate (5,10-CH2-H4PteGlu5), although in the absence of H4PteGlu5, formaldehyde is produced. Single turnover studies using substrate levels of the enzyme (2.3 microM) showed pseudo-first-order kinetics, with apparent first-order rate constants of 0.084 and 0.14 s-1 at 23 and 48.3 microM dimethylglycine, respectively, for dimethylglycine dehydrogenase and 0.065 s-1 at 47.3 microM sarcosine for sarcosine dehydrogenase. The rates were identical in the absence or presence of bound tetrahydropteroylglutamate (H4PteGlu). Titration of the enzymes with substrate under anaerobic conditions did not disclose the presence of an intermediate semiquinone. The effect of dimethylglycine concentration upon the rate of the dimethylglycine dehydrogenase reaction under aerobic conditions showed nonsaturable kinetics suggesting a second low-affinity site for the substrate which increases the enzymatic rate. The Km for the high-affinity active site was 0.05 mM while direct binding for the low-affinity site could not be measured. Sarcosine and dimethylthetin are poor substrates for dimethylglycine dehydrogenase and methoxyacetic acid is a competitive inhibitor at low substrate concentrations. At high dimethylglycine concentrations, increasing the concentration of methoxyacetic acid produces an initial activation and then inhibition of dimethylglycine dehydrogenase activity. When these compounds were added in varying concentrations to the enzyme in the presence of dimethylglycine, their effects upon the rate of the reaction were consistent with the presence of a second low-affinity binding site on the enzyme which enhances the reaction rate. When sarcosine is used as the substrate for sarcosine dehydrogenase the kinetics are Michaelis-Menten with a Km of 0.5 mM for sarcosine. Also, methoxyacetic acid is a competitive inhibitor of sarcosine dehydrogenase with a Ki of 0.26 mM. In the absence of folate, substrate and product determinations indicated that 1 mol of formaldehyde and of sarcosine or glycine were produced for each mole of dimethylglycine or sarcosine consumed with the concomitant reduction of 1 mol of bound FAD.
...
PMID:Enzymatic properties of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver. 241 60

The flavoenzymes dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) contain covalently bound FAD linked via the 8 alpha-position of the isoalloxazine ring to the imidazole N(3) of a histidine residue (Cook, R. J., Misono, K. S., and Wagner, C. (1984) J. Biol. Chem. 259, 12475-12480). The flavin-peptides from tryptic digests of these two enzymes have been isolated and sequenced. Automated sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase contained 25 amino acid residues in the following sequence: Ser-Glu-Leu-Thr-Ala-Gly-Ser- Thr-Trp-His(flavin)-Ala-Ala-Gly-Leu-Thr-Thr-Tyr-Phe-His-Pro-Gly-Ile-A sn-Leu-Lys. The sequence determined for the flavin-peptide from sarcosine dehydrogenase contained 14 amino acid residues Leu-Thr-Ser-Gly-Thr-Thr-Trp-His(flavin)-Thr-Ala-Gly-Leu-Gly-Arg.
...
PMID:The amino acid sequences of the flavin-peptides of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria. 405 29

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are the folate binding proteins of rat liver mitochondria. These two enzymes contain covalently bound flavin and catalyze similar oxidative demethylation reactions (Wittwer, A. J., and Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108). Flavin-peptides have been purified from these two enzymes after proteolytic digestion by trypsin and chymotrypsin. The spectral and chromatographic properties of these flavin peptides changed after treatment with nucleotide pyrophosphatase in a manner consistent with the conversion of an FAD-peptide to an FMN-peptide. The pKa for pH-dependent fluorescence quenching of the purified flavin-peptides was not affected by borohydride reduction which, in conjunction with the pKa values, indicated that the flavin was covalently linked via the 8 alpha position of the isoalloxazine ring to an imidazole N(3) of a histidine residue. Peptides from both enzymes showed histidylflavin at the N terminus. Amino acid composition and sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase was His(flavin)-Ala-Ala-Gly-Leu. Amino acid composition and N-terminal analysis suggested the sequence of the flavin-peptide of sarcosine dehydrogenase was His(flavin)-(Ala, Gly,Thr)-Leu.
...
PMID:Identification of the covalently bound flavin of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria. 649 Jun 27

Electron transfer flavoprotein (ETF) is a heterodimer that contains a single equivalent of FAD and accepts electrons from nine flavoprotein dehydrogenases in the mitochondrial matrix. Human ETF was expressed in Escherichia coli using the expression vector previously employed to express Paracoccus denitrificans ETF (Bedzyk, L. A., Escudero, K. W., Gill, R. E., Griffin, K. J., and Frerman, F. E. (1993) J. Biol. Chem. 268, 20211-20217). cDNAs encoding the beta and alpha subunits of the human protein were inserted into the vector, mimicking the arrangement of the P. denitrificans genes in which coding sequences are joined by overlapping termination and initiation codons. A human ETF containing 30% P. denitrificans sequence at the amino terminus of the beta subunit was also expressed and purified. This chimeric ETF has 64% sequence identity with the human sequence in the substituted region. Kinetic constants of medium chain and short chain acyl-CoA dehydrogenases for the chimeric ETFs were slightly changed from those of human ETF; but, there are marked differences in the kinetic constants of sarcosine dehydrogenase and electron transfer flavoprotein-ubiquinone oxidoreductase with the two ETFs. Absorption spectra of the three redox states of human, chimeric, and P. denitrificans ETF flavins are identical. However, the flavin circular dichroism spectra of the three ETFs are characteristic for each species. The spectrum of the chimeric ETF has both human and P. denitrificans ETF features. The amplitude of the 436 nm band is identical to that of the of the human ETF flavin, but the amplitude of the 375 nm band is identical to that of the P. denitrificans ETF flavin. Thus, flavin in the chimeric ETF appears to be exposed to dipoles in the protein framework provided by human and bacterial sequences. These spectral data indicate that the flavin is located in the vicinity of the amino-terminal region of the beta subunit. The kinetic data suggest that the amino-terminal region of the beta subunit comprises part of the docking site for some primary dehydrogenases and electron transfer flavoprotein-ubiquinone oxidoreductase.
...
PMID:Expression and characterization of human and chimeric human-Paracoccus denitrificans electron transfer flavoproteins. 779 24

Anti-mitochondrial antibodies (anti-M7) in sera from patients with dilated cardiomyopathy and myocarditis recognize, besides mitochondrial antigens, bacterial sarcosine dehydrogenase. The common target antigen was identified as the covalently bound FAD of mitochondrial and bacterial flavoenzymes. Thus, anti-M7-positive serum reacted on Western blots exclusively with covalently flavinylated enzymes. The antigenic specificity of anti-M7 sera was reproduced by an antiserum raised in rabbits with 6-hydroxy-D-nicotine oxidase. The heart mitochondrial membrane antigen recognized by anti-M7 serum was identified as the flavoprotein subunit of succinate dehydrogenase, the antigens in rat liver mitochondrial matrix as the flavoenzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase. Anti-M7 serum contained a specific anti-flavoenzyme antibody fraction. Nanomolar concentrations of FAD and riboflavin inhibited the immune reaction on Western blots and in ELISA, and incubation with FAD-agarose depleted the anti-M7 activity of the serum. N-terminally deleted dimethylglycine dehydrogenase proteins were only immunoprecipitated by anti-M7 sera when the FAD was covalently incorporated. An affinity constant (KD) of 10(-8) M was established for the anti-flavoenzyme antibodies by competitive ELISA. Of patients with cardiomyopathy and myocarditis, 36% and 25%, respectively, were anti-flavoenzyme-positive by Western blot and ELISA, but only two of 15 patients with other heart diseases and none of 50 healthy controls.
...
PMID:Anti-mitochondrial antibodies in patients with dilated cardiomyopathy (anti-M7) are directed against flavoenzymes with covalently bound FAD. 952 96

Sarcosine dehydrogenase (SarDH) is a mitochondrial flavoenzyme involved in the oxidative degradation of choline to glycine. The absence of SarDH activity in humans is genetically transmitted and is the cause of an amino acid metabolism disorder called sarcosinemia. Tryptic fragments of the purified enzyme from rat liver were subjected to Edman degradation and the sequences obtained were used to clone the cDNA encoding the full length protein. The deduced amino acid sequence of SarDH shares an overall similarity of 47% with dimethylglycine dehydrogenase (Me2GlyDH), another flavoenzyme involved in the mitochondrial choline catabolism with a similar FAD-binding domain. Covalent binding of FAD to SarDH was demonstrated by the observation of strong fluorescence at 530 nm under excitation at 450 nm of the enzyme immunoprecipitated under denaturing conditions from liver extracts. The localization of SarDH immunoreactivity in the mitochondrial matrix was confirmed by Western-blot analysis of purified mitochondrial fractions. Finally, the tissue distribution of SarDH was investigated by Northern-blot analysis of total RNA and Western-blot analysis of total protein from several rat tissues. A strong expression in the liver, but also in the lung, pancreas, kidney, thymus, and oviduct was observed. We therefore suggest that the enzymes of the choline catabolism pathway are important also for metabolism in nonhepatic tissues.
...
PMID:Molecular cloning and tissue distribution of rat sarcosine dehydrogenase. 983 43

Enteroviruses, the most common cause of acute myocarditis, are also supposed aetiological agents of dilated cardiomyopathy. Autoantibodies (anti-M7; Klein & Berg, Clin Exp Immunol 1990; 58:283-92) directed against flavoproteins with covalently bound flavin (alphaFp-Ab; Otto et al., Clin Exp Immunol 1998; 111:541-2) are detected in up to 30% of sera of patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM). Mice inoculated with a myocarditic variant of coxsackievirus B3 (CVB3) were employed to study the occurrence of serum alphaFp-Ab following viral infection. The presence of alphaFp-Ab was analysed by Western blotting with the flavoprotein antigens 6-hydroxy-D-nicotine oxidase (6HDNO) and sarcosine oxidase (SaO). Of 10 sera from CVB3-infected mice, five showed a strong reaction with both antigens. The sera were reactive also to the mitochondrial covalently flavinylated proteins dimethylglycine dehydrogenase and sarcosine dehydrogenase. Sera of non-infected mice did not react with these antigens. A 6HDNO mutant protein with non-covalently bound FAD no longer reacted on Western blots with sera of CVB3-infected mice. Preincubation with FAD abolished or reduced the reaction of the sera with the 6HDNO antigen. At 2 weeks p.i. the alphaFp-Ab were of the IgM and IgG isotypes, at 7 and 9 weeks p.i. of the IgG isotype. The sera of CVB3-infected mice reproduced closely the antigenic specificity of the anti-M7 sera of patients, lending further support to the role of coxsackieviruses in the pathogenesis of IDCM.
...
PMID:Coxsackievirus B3 infection induces anti-flavoprotein antibodies in mice. 1112 47

Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase-like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as gamma-aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating gamma-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild-type enzyme. A Km of 140 microm and a kcat of 800 s(-1) was determined when gamma-N-methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200-fold slower than gamma-N-methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the gamma-N-methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation.
...
PMID:A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1. 1560 55

The crystal structure of heterotetrameric sarcosine oxidase (TSOX) from Pseudomonas maltophilia has been determined at 1.85 A resolution. TSOX contains three coenzymes (FAD, FMN and NAD+), four different subunits (alpha, 103 kDa; beta, 44 kDa; gamma, 21 kDa; delta, 11 kDa) and catalyzes the oxidation of sarcosine (N-methylglycine) to yield hydrogen peroxide, glycine and formaldehyde. In the presence of tetrahydrofolate, the oxidation of sarcosine is coupled to the formation of 5,10-methylenetetrahydrofolate. The NAD+ and putative folate binding sites are located in the alpha-subunit. The FAD binding site is in the beta-subunit. FMN is bound at the interface of the alpha and beta-subunits. The FAD and FMN rings are separated by a short segment of the beta-subunit with the closest atoms located 7.4 A apart. Sulfite, an inhibitor of oxygen reduction, is bound at the FMN site. 2-Furoate, a competitive inhibitor with respect to sarcosine, is bound at the FAD site. The sarcosine dehydrogenase and 5,10-methylenetetrahydrofolate synthase sites are 35 A apart but connected by a large internal cavity (approximately 10,000 A3). An unexpected zinc ion, coordinated by three cysteine and one histidine side-chains, is bound to the delta-subunit. The N-terminal half of the alpha subunit of TSOX (alphaA) is closely similar to the FAD-binding domain of glutathione reductase but with NAD+ replacing FAD. The C-terminal half of the alpha subunit of TSOX (alphaB) is similar to the C-terminal half of dimethylglycine oxidase and the T-protein of the glycine cleavage system, proteins that bind tetrahydrofolate. The beta-subunit of TSOX is very similar to monomeric sarcosine oxidase. The gamma-subunit is similar to the C-terminal sub-domain of alpha-TSOX. The delta-subunit shows little similarity with any PDB entry. The alphaA domain/beta-subunit sub-structure of TSOX closely resembles the alphabeta dimer of L-proline dehydrogenase, a heteroctameric protein (alphabeta)4 that shows highest overall similarity to TSOX.
...
PMID:Heterotetrameric sarcosine oxidase: structure of a diflavin metalloenzyme at 1.85 A resolution. 1682 Jan 68

Formaldehyde is extremely toxic reacting with proteins to crosslinks peptide chains. Formaldehyde is a metabolic product in many enzymatic reactions and the question of how these enzymes are protected from the formaldehyde that is generated has largely remained unanswered. Early experiments from our laboratory showed that two liver mitochondrial enzymes, dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SDH) catalyze oxidative demethylation reactions (sarcosine is a common name for monomethylglycine). The enzymatic products of these enzymes were the demethylated substrates and formaldehyde, produced from the removed methyl group. Both DMGDH and SDH contain FAD and both have tightly bound tetrahydrofolate (THF), a folate coenzyme. THF binds reversibly with formaldehyde to form 5,10-methylene-THF. At that time we showed that purified DMGDH, with tightly bound THF, reacted with formaldehyde generated during the reaction to form 5,10-methylene-THF. This effectively scavenged the formaldehyde to protect the enzyme. Recently, post-translational modifications on histone tails have been shown to be responsible for epigenetic regulation of gene expression. One of these modifications is methylation of lysine residues. The first enzyme discovered to accomplish demethylation of these modified histones was histone lysine demethylase (LSD1). LSD1 specifically removes methyl groups from di- and mono-methylated lysines at position 4 of histone 3. This enzyme contained tightly bound FAD and the products of the reaction were the demethylated lysine residue and formaldehyde. The mechanism of LSD1 demethylation is analogous to the mechanism previously postulated for DMGDH, i.e. oxidation of the N-methyl bond to the methylene imine followed by hydrolysis to generate formaldehyde. This suggested that THF might also be involved in the LSD1 reaction to scavenge the formaldehyde produced. Our hypotheses are that THF is bound to native LSD1 by analogy to DMGDH and SDH and that the bound THF serves to protect the FAD class of histone demethylases from the destructive effects of formaldehyde generation by formation of 5,10-methylene-THF. We present pilot data showing that decreased folate in livers as a result of dietary folate deficiency is associated with increased levels of methylated lysine 4 of histone 3. This can be a result of decreased LSD1 activity resulting from the decreased folate available to scavenge the formaldehyde produced at the active site caused by the folate deficiency. Because LSD1 can regulate gene expression this suggests that folate may play a more important role than simply serving as a carrier of one-carbon units and be a factor in other diseases associated with low folate.
...
PMID:Folate deficiency affects histone methylation. 2688 Jun 41


1

---