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

Abstract

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...

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

“…Recent hydrogen-deuterium exchange analysis of NpmA-SAM/SAH complexes suggests the potential for allosteric communication between the ␤2/3 linker 30S binding surface, the ␤5/6 linker, and the cosubstrate binding pocket between them (25). NpmA interaction with cosubstrate was also previously speculated to be influenced by 30S substrate binding (20,22), based in part on the observation that substitution of ␤6/7 linker residue S195 decreases SAM/SAH affinity by Ͼ50-fold, yet the variant enzyme is still capable of conferring wild-type-level resistance. In addition to a direct hydrogen bond made by S195 to the SAM carboxylate group, a local 30S binding-induced conformational change also repositions the L196 backbone to directly interact with SAM.…”

“…S4). These deletions may be structurally tolerated given their location on the protein surface and the observed structural variability observed in these linker regions in NpmA and related enzymes (19)(20)(21).…”

“…5A). As this ␣-helical ␤5/6 linker structure appears to be unique to NpmA among the known m 1 A1408 methyltransferase structures (19)(20)(21)(22), we individually replaced two central residues, A148 and E149, with proline to test whether disrupting the ␣-helix would impact NpmA activity. However, both variants conferred wild-type MICs ( Table 1), suggesting that the precise structure formed by the ␤5/6 linker is not critical for NpmA activity.…”

Ligand-mediated changes in conformational dynamics of NpmA: implications for ribosomal interactions

“…All samples were kept at 20 °C and the exchange reaction was quenched after 0.5, 1, 2, 5 and 10 min using 40 μl ice-cold buffer containing 0.1% TFA and 2.5 M guanidinium hydrochloride; this addition brought the final pH read of the quenched sample to 2.5. Besides the shorter deuterium labeling times, maximum time of 10 min chosen was suitable as substantial methylation levels have been shown previously at 10-minutes in an in vitro methylation assay16. This quenched sample (50 μl) was then injected into a chilled nanoUPLC sample manager17 and passed at a flow rate of 100 μl/min through a 2.1 × 30-mm immobilized pepsin column (Poroszyme, ABI, Foster City, CA) in a solution of 0.1% (v/v) formic acid in water.…”

Substrate recognition by the Pseudomonas aeruginosa EF-Tu methyltransferase EftM

“…EftM PAO1 / EftM HM4 (pColdII vector) and EF-Tu (pJP04, pDEST14-N-His6tufB) for recombinant protein expression in Escherichia coli or P. aeruginosa were previously reported (9). Site-directed mutagenesis of EftM HM4 and EF-Tu were performed using a mega-primer whole-plasmid PCR approach (34,35), and the variant proteins expressed from the same parent vectors as the wild-type proteins. A construct for expression of tag-free EF-Tu, was generated by PCR amplification and subcloning the P. aeruginosa PAO1 EF-Tu (tufB) coding sequence from plasmid pJP04 (9) into the pE-SUMO vector (LifeSensors).…”