The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein.
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This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an SN2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center. (+info)
Phospholipid-assisted refolding of an integral membrane protein. Minimum structural features for phosphatidylethanolamine to act as a molecular chaperone.
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Escherichia coli-derived phosphatidylethanolamine (PE) or PE with fully saturated fatty acids was able to correct in vitro a defect in folding in the lipid-dependent epitope 4B1 of lactose permease (LacY) resulting from in vivo assembly in the absence of PE. PE plasmalogen, PE with two unsaturated fatty acids, and lyso-PE, which all do not favor bilayer organization, did not support proper refolding. Proper refolding occurred when these latter lipids were mixed with a bilayer-forming lipid (phosphatidylglycerol), which alone could not support refolding. L-Phosphatidylserine (PS; natural diastereomer) did support proper refolding. PE derivatives of increasing degrees of methylation were progressively less effective in supporting refolding, with phosphatidylcholine being completely ineffective. Therefore, the properties of nonmethylated aminophospholipids capable of organization into a bilayer configuration are essential for the recovery of the native state of epitope 4B1 after misassembly in vivo in the absence of PE. Neither D-PS (sn-glycero-1-phosphate backbone) nor P-D-S (D-serine in the head group) is competent in supporting proper refolding unless used in binary mixtures with phosphatidylglycerol. The detailed characterization of phospholipid-assisted refolding reported here further supports a specific rather than nonspecific role for PE in structural maturation of lactose permease in vivo (Bogdanov, M., and Dowhan, W. (1998) EMBO J. 17, 5255-5264). (+info)
Structural properties of DNA:RNA duplexes containing 2'-O-methyl and 2'-S-methyl substitutions: a molecular dynamics investigation.
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The physical properties of a DNA:RNA hybrid sequence d(CCAACGTTGG)*(CCAACGUUGG) with modifications at the C2'-positions of the DNA strand by 2'-O-methyl (OMe) and 2'-S-methyl (SMe) groups are studied using computational techniques. Molecular dynamics simu-lations of SMe_DNA:RNA, OMe_DNA:RNA and standard DNA:RNA hybrids in explicit water indicate that the nature of the C2'-substituent has a significant influence on the macromolecular conformation. While the RNA strand in all duplexes maintains a strong preference for C3'-endo sugar puckering, the DNA strand shows considerable variation in this parameter depending on the nature of the C2'-substituent. In general, the preference for C3'-endo puckering follows the following trend: OMe_DNA>DNA>SMe_DNA. These results are further corroborated using ab initio methods. Both gas phase and implicit solvation calculations show the C2'-OMe group stabilizes the C3'-endo conformation while the less electronegative SMe group stabilizes the C2'-endo conformation when compared to the standard nucleoside. The macromolecular conformation of these nucleic acids also follows an analogous trend with the degree of A-form character decreasing as OMe_DNA:RNA>DNA:RNA>SMe_DNA:RNA. A structural analysis of these complexes is performed and compared with experimental melting point temper-atures to explain the structural basis to improved binding affinity across this series. Finally, a possible correlation between RNase H activity and conformational changes within the minor groove of these complexes is hypothesized. (+info)
Isolation and characterization of a novel Forssman active acidic glycosphingolipid with branched isoglobo-, ganglio-, and neolacto-series hybrid sugar chains.
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Equine kidney and spleen contain a Forssman active glycosphingolipid, and the structure of this glycolipid has been reported to be that of a globopentaosylceramide (GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1, 4Galbeta-1,4Glcbeta-1,1'Ceramide). We found that equine kidney contains several other anti-Forssman antibody-reactive glycosphingolipids. One of these acidic Forssman active glycosphingolipids was isolated and characterized by means of NMR, mass spectrometry, permethylation studies, and TLC-immunostaining. This glycolipid contains three moles of galactose, one mole of glucose, three moles of N-acetylgalactosamine, one mole of N-acetylglucosamine, and one mole of N-acetylneuraminic acid, and is stained on TLC with anti-Forssman antibodies and anti-GM2 ganglioside antibodies. HOHAHA and ROESY experiments and permethylation studies showed this glycolipid oligosaccharide to be branched at the innermost galactose; one chain has an isoglobo structure with a terminal Forssman disaccharide and the other chain is branched through the linkage of N-acetylglucosaminebeta-1,6 to the inner galactose. The nonreducing end of the GM2 trisaccharide is linked to this glucosamine. The structure of the oligosaccharide of the glycolipid presented here is a novel type, having branched isoglobo-, ganglio-, and neolacto-series oligosaccharides. Mass spectrometric analyses indicated the ceramide moiety of the glycolipid to be composed predominantly of hydroxy fatty acids (C20:0, C22:0, C23:0, C24:0, and C25:0) and hydroxysphinganine. GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1,3[GalNAcbet a-1, 4(NeuAcalpha-2,3)Galbeta-1,4GlcNAcbeta-1,6]Galbeta+ ++-1,4Glcbeta-1, 1'Ceramide (+info)
Unusual sites of arginine methylation in Poly(A)-binding protein II and in vitro methylation by protein arginine methyltransferases PRMT1 and PRMT3.
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Arginine methylation is a post-translational modification found mostly in RNA-binding proteins. Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to contain NG, NG-dimethylarginine at 13 positions in its amino acid sequence. Two additional arginine residues were partially methylated. Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein. These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation. Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli were in vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, with S-adenosyl-L-methionine as the methyl group donor. Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites. (+info)
RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: Tests of transcription factor involvement in nucleolar dominance.
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In interspecific hybrids or allopolyploids, often one parental set of ribosomal RNA genes is transcribed and the other is silent, an epigenetic phenomenon known as nucleolar dominance. Silencing is enforced by cytosine methylation and histone deacetylation, but the initial discrimination mechanism is unknown. One hypothesis is that a species-specific transcription factor is inactivated, thereby silencing one set of rRNA genes. Another is that dominant rRNA genes have higher binding affinities for limiting transcription factors. A third suggests that selective methylation of underdominant rRNA genes blocks transcription factor binding. We tested these hypotheses using Brassica napus (canola), an allotetraploid derived from B. rapa and B. oleracea in which only B. rapa rRNA genes are transcribed. B. oleracea and B. rapa rRNA genes were active when transfected into protoplasts of the other species, which argues against the species-specific transcription factor model. B. oleracea and B. rapa rRNA genes also competed equally for the pol I transcription machinery in vitro and in vivo. Cytosine methylation had no effect on rRNA gene transcription in vitro, which suggests that transcription factor binding was unimpaired. These data are inconsistent with the prevailing models and point to discrimination mechanisms that are likely to act at a chromosomal level. (+info)
An inverted repeat triggers cytosine methylation of identical sequences in Arabidopsis.
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The Wassilewskija (WS) strain of Arabidopsis has four PAI genes at three sites: an inverted repeat at one locus plus singlet genes at two unlinked loci. These four genes are methylated over their regions of DNA identity. In contrast, the Columbia (Col) strain has three singlet PAI genes with no methylation. To test the hypothesis that the WS inverted repeat locus triggers methylation of unlinked identical sequences, we introduced this locus into the Col background by genetic crosses. The inverted repeat induced de novo methylation of all three unmethylated Col PAI genes, with methylation efficiency varying with the position of the target locus. These results, plus results with inverted repeat transgenes, show that methylation is communicated by a DNA/DNA pairing mechanism. (+info)
Cysteine carboxyl O-methylation of human placental 23 kDa protein.
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C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling. (+info)