Expansion of the aminoglycoside-resistance 16S rRNA (m1A1408) methyltransferase family: Expression and functional characterization of four hypothetical enzymes of diverse bacterial origin

Self Cite

The pathogen-associated 16S rRNA methyltransferase NpmA catalyzes m 1 A1408 modification to block the action of structurally diverse aminoglycoside antibiotics. Here, we describe the development of a fluorescence polarization binding assay and its use, together with complementary functional assays, to dissect the mechanism of NpmA substrate recognition. These studies reveal that electrostatic interactions made by the NpmA ␤2/3 linker collectively are critical for docking of NpmA on a conserved 16S rRNA tertiary surface. In contrast, other NpmA regions (␤5/␤6 and ␤6/␤7 linkers) contain several residues critical for optimal positioning of A1408 but are largely dispensable for 30S binding. Our data support a model for NpmA action in which 30S binding and adoption of a catalytically competent state are distinct: docking on 16S rRNA via the ␤2/3 linker necessarily precedes functionally critical 30S substrate-driven conformational changes elsewhere in NpmA. This model is also consistent with catalysis being completely positional in nature, as the most significant effects on activity arise from changes that impact binding or stabilization of the flipped A1408 conformation. Our results provide a molecular framework for aminoglycoside resistance methyltransferase action that may serve as a functional paradigm for related enzymes and a starting point for development of inhibitors of these resistance determinants.KEYWORDS RNA binding proteins, aminoglycosides, antibiotic resistance, methyltransferase, rRNA modification, ribosomes A minoglycosides are potent antimicrobial agents used for clinical treatment of life-threatening infections of both Gram-positive and Gram-negative bacteria and are also used routinely for both veterinary and growth promotion applications in agricultural settings (1, 2). Most aminoglycosides bind 16S rRNA helix 44 (h44) to induce conformational changes in the universally conserved nucleotides A1492 and A1493 in the ribosome decoding center. As a result, the bacterial ribosome is rendered unable to accurately discern cognate mRNA-tRNA pairing, thus impairing translational fidelity (3-9). More recent evidence has also suggested an additional 23S rRNA binding site for some aminoglycosides which disrupts intersubunit bridge B2, impacting a ribosomal conformational change required during elongation (10).Both aminoglycoside-producing and human-pathogenic bacteria can achieve high levels of resistance to aminoglycosides by reducing drug permeability or increasing efflux from the cell, enzymatic chemical modification of the drug, or mutation or chemical modification of the aminoglycoside binding site (4,11,12). In particular, S-adenosyl-L-methionine (SAM)-dependent 16S rRNA methyltransferases are the predominant resistance mechanism found in aminoglycoside-producing bacteria and are an increasing clinical concern with their continued emergence in major human patho-

Section: Methodsmentioning

confidence: 99%

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

Vinal

Conn

2017

Self Cite

“…This recombinant Kmr protein, with no additional tags at either terminus, was robustly expressed (data not shown) and provided high-level resistance to kanamycin and apramycin but not to gentamicin in E. coli BL21(DE3) ( Table 1). This resistance phenotype corresponds to that of other 16S rRNA (m 1 A1408) aminoglycoside resistance-conferring methyltrans-ferases (Table 1) (10,27) and suggests that Kmr likely also methylates 16S rRNA on A1408.…”

Section: Resultsmentioning

confidence: 99%

30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr

Savic

Sunita

Zelinskaya

et al. 2015

Self Cite

bMethylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core ␤-strands 6 and 7, which forms part of the S-adenosyl-L-methionine (SAM) binding pocket and contributes to base flipping by the m 1 A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate. Gene pools in soil and aquatic microbial communities represent largely unexplored reservoirs of antibiotic resistance (1, 2). An analysis of the resistance mechanisms present in these communities may reveal new insights into the origins, activities, and potential for mobilization of antibiotic resistance determinants to pathogenic bacterial populations. In particular, myxobacteria of the genus Sorangium have become a major source of secondary metabolites over the last several decades alongside the actinobacteria and fungi (3, 4). The model strain Sorangium cellulosum So ce56 possesses a 13.1-Mb genome with 17 gene clusters for secondary metabolites (5). Associated with this tremendous biosynthetic power is a high resistance potential against multiple antibiotics. For example, all Sorangium strains can grow in the presence of exceptionally high concentrations of kanamycin through the action of a single, specific aminoglycoside resistance-conferring 16S rRNA methyltransferase, Kmr (6).In common with most aminoglycosides, kanamycin binds within helix (h) 44 of the 16S rRNA in the bacterial small (30S) ribosomal subunit and induces errors in mRNA decoding (7-9). Aminoglycoside-producing bacteria typically protect themselves from self-intoxication by expression of aminoglycoside resistance-conferring methyltransferases that modify the drug binding site. Two subfamilies of 16S rRNA methyltransferases introduce distinct 16S rRNA modifications at N7 of guanosine 1405 (m 7 G1405) or N1 of adenosine 1408 (m 1 A140...

“…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-A1408G subunits. Additionally, we observed no difference in NpmA modification of wild-type 30S isolated from the ⌬7prrn strain and E. coli MRE600, typically used in these assays (12,13). Second, we isolated wild-type 30S subunits from E. coli BL21(DE3) cells grown without and with KamB overexpression, and tested the activity of NpmA and KamB against these unmodified and m 1 A1408 premodified substrates using the [ 3 H]SAM assay (Fig.…”

mentioning

confidence: 99%

“…Neither NpmA nor KamB could incorporate 3 H into subunits previously modified with m 1 A1408, demonstrating that NpmA does not possess activity at an alternative 16S rRNA nucleotide outside the region examined in our RT assay. From these experiments, we conclude that NpmA methylates 30S-A1408G subunits at nucleotide 1408 and thus possesses dual A1408/G1408 modification activity.To address the question of whether this novel activity is unique to NpmA, we tested the ability of Kmr and five other 16S rRNA (m 1 A1408) family members (13,14) to modify the 30S-A1408G subunits. Although all enzymes robustly modified wild-type subunits in vitro, none possessed catalytic activity with the 30S-A1408G substrate (Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Zelinskaya

Witek

Conn

2015

Self Cite

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-...