Photoaffinity labeling and mutational analysis of 24-C-sterol methyltransferase defines the AdoMet binding site.
Photolabeling and site-directed mutagenesis were performed on recombinant Saccharomyces cerevisiae 24-C-sterol methyltransferase (SMT) to elucidate the location and role of active site residues involved in AdoMet binding and catalysis. Bioinformatic analysis of the SMT revealed a ten amino acid segment, conserved between L124 and P133, associated with the Rossmann-like fold belonging to AdoMet-dependent methyltransferases. Irradiation of the SMT in the presence of [methyl-3H3]AdoMet directly photolabeled the protein. The specificity of photolabeling was demonstrated by inactivation experiments with structural analogs of AdoMet, including sinefungin. Trypsin digestion of the [methyl-3H3]AdoMet photolabeled Erg6p afforded a single radioactive band in SDS-PAGE gel of 4 kDa. HPLC purification of this material generated a single radioactive fraction. The corresponding 3H-AdoMet-peptide adduct was subjected to Edman sequencing and the first fifteen residues gave a sequence Gly120-Asp-Leu-Val-Leu-Asp-Val-Gly-Cys-Gly-Val-Gly-Gly-Pro-Ala134 that contained the predicted AdoMet binding site. Amino acid residues in the tryptic digest fragment considered to bind covalently with the AdoMet at Asp125, Cys128, Pro133 and Tyr153 were replaced with leucine and analyzed kinetically and by photolabeling inactivation experiments. The results indicate that one or both of Cys128 and Pro133 are covalently bound to AdoMet.
Jayasimha
Pruthvi
P
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.
Nes
W David
WD
D001665:Binding Sites; D004252:DNA Mutational Analysis; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015335:Molecular Probes / Q000032:analysis* / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010447:Peptide Hydrolases / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry*; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment
D015335:Molecular Probes; D019920:Photoaffinity Labels; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; D010447:Peptide Hydrolases