Publications

• A novel automethylation reaction in the Aspergillus nidulans LaeA protein generates S-methylmethionine.

  Patananan A.N., Palmer J.M., Garvey G.S., Keller N.P., Clarke S.G.

  The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that can adapt to their environment by producing various secondary metabolites, including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are ... >> More

  The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that can adapt to their environment by producing various secondary metabolites, including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA in all pathogenic fungi examined to date show decreased virulence coupled with reduced secondary metabolism. Although the amino acid sequence of LaeA contains the motifs characteristic of seven-β-strand methyltransferases, a methyl-accepting substrate of LaeA has not been identified. In this work we did not find a methyl-accepting substrate in Aspergillus nidulans with various assays, including in vivo S-adenosyl-[methyl-(3)H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self-methylate. Amino acid hydrolysis of radioactively labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine 207. However, in vivo complementation showed that methionine 207 is not required for the biological function of LaeA. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S-adenosylmethionine but also provide new insights into the physiological function of LaeA. << Less

  J. Biol. Chem. 288:14032-14045(2013) [PubMed] [EuropePMC]

• Purification and characterization of enzymes from Euglena gracilis that methylate methionine and arginine residues of cytochrome c.

  Farooqui J.Z., Tuck M., Paik W.K.

  Two forms of cytochrome c-specific methyltransferases from Euglena gracilis were purified approximately 100- and 50-fold, respectively, using DEAE-cellulose and gel-filtration chromatography. The methylation product of enzyme I was identified as S-methylmethionine and that of enzyme II as NG-monom ... >> More

  Two forms of cytochrome c-specific methyltransferases from Euglena gracilis were purified approximately 100- and 50-fold, respectively, using DEAE-cellulose and gel-filtration chromatography. The methylation product of enzyme I was identified as S-methylmethionine and that of enzyme II as NG-monomethylarginine. Both enzymes were located in the cytosol and exhibit maximum activity at pH 7.0. Among the various proteins tested as substrates, the enzymes were highly specific toward cytochrome c. Various types of histones, in particular, were not modified by either enzyme. The molecular weights of enzyme I and II were 28,000 and 36,000, respectively. Various S-adenosyl-L-homocysteine analogs were tested for their inhibitory activity toward the enzymes. Only the D- and L-isomers of S-adenosylhomocysteine and sinefungin were significantly inhibitory. The Ki values for S-adenosyl-L-homocysteine were 8.13 X 10(-6) and 1.17 X 10(-5) M for enzyme I and II, respectively. Two-dimensional peptide mapping revealed the methylation site of enzyme I to be the methionine residue at position 65 while that of enzyme II to be the arginine residue at position 38. The methylation of either methionine or arginine residues by enzyme I and II, respectively, lowers the isoelectric point (pI) of cytochrome c: 9.23, 9.33, and 10.06 for S-methylmethionine-, NG-monomethylarginine-modified, and unmodified cytochrome c, respectively. << Less

  J Biol Chem 260:537-545(1985) [PubMed] [EuropePMC]

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