title: Structural and functional plasticity of antibiotic resistance nucleotidylyltransferases revealed by molecular characterization of lincosamide nucleotidylyltransferases lnu(A) and lnu(D).
abstract: One of the main mechanisms of resistance to lincosamide and aminoglycoside antibiotics is their inactivation by O-nucleotidylyltransferases (NTases). Significant sequence variation of lincomycin nucleotidylyltransferase (Lnu) and aminoglycoside nucleotidylyltransferase (ANT) enzymes plus lack of detailed information about the molecular basis for specificity of these enzymes toward chemically distinct antibiotic scaffolds hinders development of a general strategy to curb this resistance mechanism. We conducted an extensive sequence analysis identifying 129 putative antibiotic NTases constituting six distinct subfamilies represented by Lnu(A), Lnu(B), Lnu(C), Lnu(D), Lnu(F)/(G) plus ANT(2") enzymes. Since only the Lnu(B) enzyme has been previously studied in detail, we biochemically characterized the Lnu(A) and Lnu(D) enzymes, with the former representing the most sequence distinct Lnu ortholog. We also determined the crystal structure of the Lnu(A) enzyme in complex with a lincosamide. These data suggested that, while sharing the N-terminal nucleotidylyltransferase domain, the groups of antibiotic NTases feature structurally distinct C-terminal domains (CTDs) adapted to accommodate antibiotics. Comparative structural analysis among antibiotic NTases rationalized their specificity toward lincosamides versus aminoglycosides through active-site plasticity, which allows retention of general catalytic activity while accepting alterations at multiple, specific positions contributed by both domains. Based on this structural analysis, we suggest that antibiotic NTases evolved from an ancestral nucleotidylyltransferase along independent paths according to the identified groups, characterized by structural changes in the active site and recruitment of structurally diverse CTDs. These data show the complexity of enzyme-driven antibiotic resistance and provide a basis for broadly active inhibitors by identifying the key unifying features of antibiotic NTases.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada M5G 1L6; Center for Structural Genomics of Infectious Diseases (CSGID).
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Stogios
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada M5G 1L6; Center for Structural Genomics of Infectious Diseases (CSGID).
authors.forename: Elena
authors.initials: E
authors.lastname: Evdokimova
authors.affiliation: M. G. DeGroote Institute for Infectious Disease Research, McMaster University, MDCL 2301, 1280 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
authors.forename: Mariya
authors.initials: M
authors.lastname: Morar
authors.affiliation: M. G. DeGroote Institute for Infectious Disease Research, McMaster University, MDCL 2301, 1280 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
authors.forename: Kalinka
authors.initials: K
authors.lastname: Koteva
authors.affiliation: M. G. DeGroote Institute for Infectious Disease Research, McMaster University, MDCL 2301, 1280 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
authors.forename: Gerard D
authors.initials: GD
authors.lastname: Wright
authors.affiliation: Unité des Agents Antibacteriens, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Courvalin
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada M5G 1L6; Center for Structural Genomics of Infectious Diseases (CSGID). Electronic address: alexei.savchenko@utoronto.ca.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D016000:Cluster Analysis; D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial; D055231:Lincosamides / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D055231:Lincosamides; D009713:Nucleotidyltransferases; C057535:lincosaminide O-nucleotidyltransferase
keywords: aminoglycoside; antibiotic resistance; crystal structure; lincosamide; nucleotidylyltransferase