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Structural and functional characterization of CFE88: Evidence that a conserved and essential bacterial protein is a methyltransferase
Author(s) -
Constantine Keith L.,
Krystek Stanley R.,
Healy Matthew D.,
Doyle Michael L.,
Siemers Nathan O.,
Thanassi Jane,
Yan Ning,
Xie Dianlin,
Goldfarb Valentina,
Yanchunas Joseph,
Tao Li,
Dougherty Brian A.,
Farmer Bennett T.
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051389605
Subject(s) - methyltransferase , threading (protein sequence) , protein structure , active site , dna methyltransferase , protein domain , protein secondary structure , conserved sequence , biochemistry , chemistry , stereochemistry , sequence alignment , peptide sequence , biology , crystallography , enzyme , gene , methylation
CFE88 is a conserved essential gene product from Streptococcus pneumoniae . This 227‐residue protein has minimal sequence similarity to proteins of known 3Dstructure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side‐chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N‐terminal domain with 163 residues and a C‐terminal domain with 64 residues. The C‐terminal domain is primarily helical, while the N‐terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X‐ray structure of ErmC′ methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S‐adenosyl‐L‐homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N‐terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.