title: Structural and functional characterization of CFE88: evidence that a conserved and essential bacterial protein is a methyltransferase.
abstract: CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. 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.
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543. keith.constantine@bms.com.
authors.forename: Keith L
authors.initials: KL
authors.lastname: Constantine
authors.forename: Stanley R
authors.initials: SR
authors.lastname: Krystek
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Healy
authors.forename: Michael L
authors.initials: ML
authors.lastname: Doyle
authors.forename: Nathan O
authors.initials: NO
authors.lastname: Siemers
authors.forename: Jane
authors.initials: J
authors.lastname: Thanassi
authors.forename: Ning
authors.initials: N
authors.lastname: Yan
authors.forename: Dianlin
authors.initials: D
authors.lastname: Xie
authors.forename: Valentina
authors.initials: V
authors.lastname: Goldfarb
authors.forename: Joseph
authors.initials: J
authors.lastname: Yanchunas
authors.forename: Li
authors.initials: L
authors.lastname: Tao
authors.forename: Brian A
authors.initials: BA
authors.lastname: Dougherty
authors.forename: Bennett T
authors.initials: BT
authors.lastname: Farmer
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D008780:Methyltransferases / Q000737:chemistry*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D008780:Methyltransferases