The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs

Abbott Laboratories, D46Y-AP 10, 100 Abbott Park Road, Abbott Park, IL, 60064, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 07/1999; 289(2):277-91. DOI: 10.1006/jmbi.1999.2788
Source: PubMed


The rRNA methyltransferase ErmC' transfers methyl groups from S -adenosyl-l-methionine to atom N6 of an adenine base within the peptidyltransferase loop of 23 S rRNA, thus conferring antibiotic resistance against a number of macrolide antibiotics. The crystal structures of ErmC' and of its complexes with the cofactor S -adenosyl-l-methionine, the reaction product S-adenosyl-l-homocysteine and the methyltransferase inhibitor Sinefungin, respectively, show that the enzyme undergoes small conformational changes upon ligand binding. Overall, the ligand molecules bind to the protein in a similar mode as observed for other methyltransferases. Small differences between the binding of the amino acid parts of the different ligands are correlated with differences in their chemical structure. A model for the transition-state based on the atomic details of the active site is consistent with a one-step methyl-transfer mechanism and might serve as a first step towards the design of potent Erm inhibitors.

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    • "Methyltransferases (MTases) transfer a methyl group from a donor to an acceptor, and catalyze a diverse range of substrates (small organic molecules, DNA, RNA, proteins, lipids) [27]. The rRNA MTase ErmC' methylates an adenine base in 23S rRNA, which confers resistance by obstructing the contact site for the antibiotics [28]. "
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    ABSTRACT: The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing 'duplicate' residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. 'Duplicate' residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins.
    Full-text · Article · Nov 2012 · PLoS ONE
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    • "The crystal structure of Mtf1 showed that it consists of two domains, a large α/β N-terminal domain and a smaller tetra-α helical C-terminal domain separated by a cleft (20). Structurally the N-terminal domain was closely related to a series of rRNA methyltransferases (21–24). "
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    ABSTRACT: We have characterized the mitochondrial transcription factor (Mtf1) and RNA polymerase (Rpo41) of Schizosaccharomyces pombe. Deletion mutants show Mtf1 or Rpo41 to be essential for cell growth, cell morphology and mitochondrial membrane potential. Overexpression of Mtf1 and Rpo41 can induce mitochondrial transcription. Mtf1 and Rpo41 can bind and transcribe mitochondrial promoters in vitro and the initiating nucleotides were the same in vivo and in vitro. Mtf1 is required for efficient transcription. We discuss the functional differences between Mtf1 and Rpo41 of S. pombe with Saccharomyces cerevisiae and higher organisms. In contrast to S. cerevisiae, the established model for mitochondrial transcription, S. pombe, a petite-negative yeast, resembles higher organisms that cannot tolerate the loss of mitochondrial function. The S. pombe and human mitochondrial genomes are similar in size and much smaller than that of S. cerevisiae. This is an important first step in the development of S. pombe as an alternative and complementary model system for molecular genetic and biochemical studies of mitochondrial transcription and mitochondrial–nuclear interactions. This is the first systematic study of the cellular function and biochemistry of Rpo41 and Mtf1 in S. pombe.
    Full-text · Article · Feb 2011 · Nucleic Acids Research
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    • "Because of uncertainties in the position of the S-adenosyl methionine (SAM) cofactor, this small molecule was not included during the refinement. Instead, the SAM moiety was transformed to adopt a binding mode similar to that in the ErmC′ structure (PDB entry: 1QAO8); full-atom refinement was performed under the Rosetta energy function augmented with restraints that preserved hydrogen bonding and packing interactions, analogous to those observed for ErmC′. The current unavailability of the crystallographic structure for T33 prevents us from assessing the accuracy of this small-molecule remodeling. "
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    Full-text · Article · Nov 2010 · Proteins Structure Function and Bioinformatics
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