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Structural and functional characterization of CFE88: Evidence that a conserved and essential bacterial protein is a methyltransferase

¹ Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA
² Bristol-Myers Squibb Pharmaceutical Research Institute, Pennington, New Jersey 08534, USA
³ Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492, USA

(RECEIVED February 1, 2005; FINAL REVISION March 11, 2005; ACCEPTED March 12, 2005)

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.

Keywords: bioinformatics; biophysical methods; conserved essential bacterial gene; methyltransferase; mutagenesis; nuclear magnetic resonance; protein modeling; Streptococcus pneumoniae

Abbreviations: CEG, conserved essential gene • cfe, conserved essential gene for expression • HSQC, heteronuclear single-quantum coherence • ITC, isothermal titration calorimetry • Kd, dissociation constant • HMMs, hidden Markov models • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser effect • NOESY, nuclear Overhauser effect spectroscopy • PDB, Protein Data Bank • RID, residue information data structure • rRNA, ribosomal ribonucleic acid • SAH, S-adenosyl- L-homocysteine • SAM, S-adenosyl-L-methionine • _m, mixing time • T_m, melting temperature (transition midpoint) • 2D, two-dimensional • 3D, three-dimensional • 4D, four-dimensional.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051389605.

Reprint requests to: Keith L. Constantine, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543; e-mail: keith.constantine{at}bms.com; fax: (609) 252-6030.

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