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), (B), (C), (D), (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 (NTD), the groups of antibiotic NTases feature structurally distinct C-terminal domains (CTD) adapted to accommodate antibiotics. Comparative structural analysis among antibiotic NTases rationalized their specificity toward lincosamides vs. 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 provides a basis for broadly-active inhibitors by identifying the key unifying features of antibiotic NTases.

Esterase from a cariogenic bacterium hydrolyzes dental resins

“…The gene candidates ( SMU_118c, SMU_400, SMU_643, SMU_1443c, SMU_1678 ) were PCR amplified from S. mutans UA159 genomic DNA by using designed primers (Table 1), then cloned into the p15Tv-LIC vector as previously described (empty vector was used as a control) [40], providing an N-terminal His6-tag fusion followed by a TEV protease cleavage site. The esterases were expressed in E. coli BL21 (DE3) then harvested for further protein isolation and purification [41]. Cells were resuspended in binding buffer [50 mM Hepes (pH 7.5), 100/300 mM NaCl, 10 mM imidazole and 2% glycerol (v/v)], lysed using a sonicator, and cell debris was removed via centrifugation at 30,000G.…”

Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes

“…The aac­(6 ′ )-Ig and the -Ih sequences were PCR amplified from A. haemolyticus BM2685 genomic DNA and A. baumannii plasmid pAT79 DNA, respectively, and cloned by ligase-independent cloning in the p15TV-LIC vector, which provided an N-terminal His6-tag fusion followed by a TEV protease cleavage site. Proteins were expressed natively following the protocol detailed in ref .…”

Rifampin phosphotransferase is an unusual antibiotic resistance kinase

“…Furthermore, these resistance genes have evolved from, or share common ancestors with, proto-resistance genes encoding proteins that have other cellular functions 9 . Notable examples include aminoglycoside and lincosamide nucleotidyltransferases ( ant and lnu genes), which exhibit ancestral connections to DNA polymerases 10 11 12 ; the aminoglycoside acetyltransferase ( aac(2′)-Ia ) from Providenica stuartii , which is thought to modify peptidoglycan under its native conditions 13 ; the chloramphenicol kinase that is closely related to both shikimate and adenylate kinases 14 ; and aminoglycoside/macrolide phosphotransferases that exhibit functional and structural similarities to lipid and protein kinases 15 16 . In most of these cases, due to low amino acid sequence homology, structural studies have been necessary to reveal the relationship between proto- and bona fide resistance genes.…”