Discovery and characterization of novel methyltransferase reactions and pathways for the amelioration of molecular aging in microorganisms

S-Adenosylmethionine (AdoMet) is a common cofactor used in enzymatic reactions which require a methyl-donor. Here I present novel methodologies for identifying and screening genes that have a high likelihood of catalyzing AdoMet dependent methyltransfer reactions. I then analyze two specific methyltransferases as well as a gene product linked to a protein repair methyltransferase.; Using custom and publicly available tools, a sequence-based signature is extracted from sequences of known methyltransferases and used to generate lists of putative methyltransferases from genomic sequences. My analysis suggests that AdoMet methyltransferases make up ∼1% of the open reading frames of the genomes examined. Having generated lists of putative methyltransferases, I describe methodologies for rapid screening and functional assignment of methyltransferases in Saccharomyces cerevisiae.; A detailed functional assignment of trans-aconitate methyltransferase (TMT1) is performed using mass spectral techniques. It is shown that 2-isopropylmalate is a major endogenous substrate and is suggestive of a general role of TMTI in methylene addition biochemistry, here with leucine biosynthesis and with the other known major substrate in yeast, trans -aconitate, a citric acid cycle derivative.; Next, the absence of the highly conserved protein L-isoaspartyl-O-methyltranferase (PIMT) gene is examined in S. cerevisiae. PIMT catalyses the repair of a common form of protein damage: the accumulation of isoaspartyl residues in peptide chains. I show that yeast lack a PIMT gene, lack PIMT activity and, in spite of this, also lack any significant level of isoaspartyl formation. I show that this lack of damage is apparently due to a lowered pH common in yeast growth conditions and suggest that the presence of PIMT activity might be harmful to yeast physiology by disrupting AdoMet homeostasis.; Finally, I show that the SurE gene product of prokaryotes (the reading frame of which is frequently found associated with that of PIMT) has an acid phosphatase activity. I show that the gene product in both T. maritima and P. aerophilum shows selective activity towards purine monophosphates and that the activity requires a metal cofactor. I suggest that SurE may act as a scavenging enzyme for nucleic acids thereby complementing the role of PIMT as a protein repair enzyme.