The Pathogen-Derived Aminoglycoside Resistance 16S rRNA Methyltransferase NpmA Possesses Dual m ¹ A1408/m ¹ G1408 Specificity

Abstract

Chemical modification of 16S rRNA can confer exceptionally high-level resistance to a diverse set of aminoglycoside antibiotics. Here, we show that the pathogen-derived enzyme NpmA possesses dual m 1 A1408/m 1 G1408 activity, an unexpected property apparently unique among the known aminoglycoside resistance 16S rRNA (m 1 A1408) methyltransferases. Although the biological significance of this activity remains to be determined, such mechanistic variation in enzymes acquired by pathogens has significant implications for development of inhibitors of these emerging resistance determinants. Methylation of 16S rRNA has emerged as a significant new threat to the efficacy of aminoglycoside antibiotics, particularly against Gram-negative pathogens (1), with two families of resistance methyltransferase defined by the modifications they produce, m 7 G1405 and m 1 A1408 (Fig. 1A). Structural and functional studies of enzymes from each family, and of both aminoglycoside producer and pathogenic origin, have firmly established them as class I methyltransferases (2) and have begun to reveal the molecular details of their interactions with the cosubstrate S-adenosyl-L-methionine (SAM) and the 30S subunit substrate (3-7).Despite these advances, important questions remain about the molecular mechanisms of action of these enzymes. For example, studies of the pathogen-derived 16S rRNA (m 1 A1408) methyltransferase NpmA (8) and the orthologous enzyme Kmr from Sorangium cellulosum So ce56 (9) revealed a critical but undefined role for the 30S in promoting SAM binding and/or catalysis. We therefore sought to establish a system to dissect the mechanism by which the 30S subunit regulates m 1 A1408 methyltransferase activity. To this end, we obtained the wild-type (⌬7prrn) Escherichia coli strain SQZ10 and its 16S rRNA A1408G variant (⌬7prrn-A1408G), in which all chromosomal rRNA operons are replaced by a single plasmid-borne copy (10, 11). Our expectation was that subunits isolated from the A1408G strain would allow analysis of 30S enzyme-SAM interactions in the absence of the modification reaction.We first used an established in vitro methylation assay with [ 3 H]SAM (12), to verify that neither NpmA nor KamB, a 16S rRNA (m 1 A1408) methyltransferase from the tobramycin producer Streptoalloteichus tenebrarius, had activity against the 30S-A1408G subunits. To our surprise, however, we found that NpmA, but not KamB, did in fact appear to methylate the variant 30S subunits (Fig. 1B), and we decided to examine this unexpected enzymatic activity.To identify the site of modification in 30S-A1408G, we performed two additional assays. First, reverse transcription (RT) of 16S rRNA extracted from untreated and NpmA-or KamBtreated wild-type and 30S-A1408G subunits was used to visualize the modified nucleotide ( Fig. 1C and D). Correlating with the [ 3 H]SAM assays, a strong stop corresponding to m 1 A1408 modification was observed for both enzymes with wild-type 30S, whereas only NpmA treatment resulted in a band at the equivalent position for 30S-...

Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase

“…Whatever its origin, this inherent redundancy makes NpmA capable of conferring exceptionally high-level aminoglycoside resistance to a broad range of bacterial species, even where alterations to the 16S rRNA docking surface might otherwise have reduced its ability to bind and methylate A1408. Overevolution of 30S binding affinity and the likely positional nature of catalysis by NpmA may also underpin our previous unexpected finding that, among all m 1 A1408 16S rRNA methyltransferases characterized, NpmA alone is able to similarly modify G1408 mutant 30S ribosome subunits (32). Thus, while the fundamental mechanism of 30S substrate docking via the ␤2/3 linker likely is conserved in this enzyme family, the overevolution of this interaction may be unique to the only currently identified pathogen-associated member.…”

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA

“…Like TlyA, NpmA relies heavily on recognition of complex rRNA structure, distant from the site of modification, to accomplish specific binding to its substrate (42). Further, a single direct base edge contact is made by NpmA to A1408, but the absolute importance of this interaction is unclear given that NpmA retains partial activity against ribosomes with a G1408 nucleotide (44). Similarly, TlyA contacts the nucleobase of C2144 via Ser234 located in the loop linking the sixth and seventh b-strands (b6/7 linker) of its Class I methyltransferase core fold, a region commonly associated with substrate recognition by these enzymes (34,45).…”

RNA nucleotide methylation: 2021 update

“…This is the case for resistance to various aminoglycosides conferred by SSU MTases from the ArmA/Rmt family, which methylate N7 of G1405 (Zarubica et al, 2011), by the 16S rRNA m 5 C1404-specific MTase EfmM in Enterococcus faecium (Galimand et al, 2011) and by NpmA, which modifies 16S-A1408 to m 1 A (Dunkle et al, 2014), (Kanazawa et al, 2017). NpmA may also modify G1408 to m 1 G in an additional case of dual specificity (Zelinskaya et al, 2015). Resistance to the macrolide antibiotic tylosin is conferred by two m 1 G residues in 23S rRNA formed by TlrD and RlmA I (Yakhnin et al, 2019).…”