Abstract
A new program (Multiple Motif Scanning) was developed to scan the Saccharomyces cerevisiae proteome for Class I S-adenosylmethionine-dependent methyltransferases. Conserved Motifs I, Post I, II, and III were identified and expanded in known methyltransferases by primary sequence and secondary structural analysis through hidden Markov model profiling of both a yeast reference database and a reference database of methyltransferases with solved three-dimensional structures. The roles of the conserved amino acids in the four motifs of the methyltransferase structure and function were then analyzed to expand the previously defined motifs. Fisher-based negative log statistical matrix sets were developed from the prevalence of amino acids in the motifs. Multiple Motif Scanning is able to scan the proteome and score different combinations of the top fitting sequences for each motif. In addition, the program takes into account the conserved number of amino acids between the motifs. The output of the program is a ranked list of proteins that can be used to identify new methyltransferases and to reevaluate the assignment of previously identified putative methyltransferases. The Multiple Motif Scanning program can be used to develop a putative list of enzymes for any type of protein that has one or more motifs conserved at variable spacings and is freely available (www.chem.ucla.edu/files/MotifSetup.Zip). Finally hidden Markov model profile clustering analysis was used to subgroup Class I methyltransferases into groups that reflect their methyl-accepting substrate specificity.
Highlights
A new program (Multiple Motif Scanning) was developed to scan the Saccharomyces cerevisiae proteome for Class I S-adenosylmethionine-dependent methyltransferases
Enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine to protein, nucleic acid, lipid, and small molecule substrates are widely distributed in nature and function in a variety of biological pathways including metabolic regulation, gene expression, the repair of aging biomolecules, and biosynthesis [1]
It has been estimated that about 0.6 –1.6% of genes in organisms ranging from Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and humans encode Class I methyltransferases [6]
Summary
Motif Identification Using Two Reference Groups of Known Methyltransferases—To search for putative methyltransferases, two reference groups of known methyltransferases were compiled to refine the consensus sequence at each motif. The HMM profile comparisons and the secondary sequences were used to objectively identify Motifs I, Post I, II, and III (Fig. 1 and supplemental Table 1) and generate the “yeast matrix” for scoring in the Multiple Motif Scanning program. Preference for using a particular structure from the list of homologous proteins includes, in descending order of importance, crystallization with S-adenosylhomocysteine or AdoMet, a minimal number of mutations, and derivation from a higher organism This reference group was used to generate the “crystal matrix” for scoring in the Multiple Motif Scanning program. The program is designed to first recognize the top five matches for the first sequence motif (here Motif I) in the amino acid sequences of the entire yeast or human proteomes [10] For each of these Motif I matches, the program searches each protein for the best five matches, considering both sequence and spacing, for the second sequence motif (here Motif Post I). Negative log p values with a cutoff of 20 were used to compile cluster groups using the program Biolayout [11]
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