Crystal Structure of the Nosiheptide-Resistance Methyltransferase of Streptomyces actuosus

Abstract

Nosiheptide-resistance methyltransferase (NHR) of Streptomyces actuosus is a class IV methyltransferase of the SpoU family and methylates 23S rRNA at nucleotide adenosine corresponding to A1067 in Escherichia coli. Such methylation is essential for resistance against nosiheptide, a sulfur peptide antibiotic, which is produced by the nosiheptide-producing strain, S. actuosus. Here, we report the crystal structures of NHR and NHR in complex with SAM (S-adenosyl-l-methionine) at 2.0 and 2.1 A resolution, respectively. NHR forms a functional homodimer, and dimerization is required for methyltransferase activity. The monomeric NHR is comprised of the N-terminal RNA binding domain (NTD) and the C-terminal catalytic domain (CTD). Overall, the structure of NHR suggests that the methyltransferase activity is achieved by "reading" the RNA substrate with NTD and "adding" methyl group using CTD. Comprehensive mutagenesis and methyltransferase activity assays reveal critical regions for SAM binding in CTD and loops (L1 and L3) essential for RNA recognition in NTD. Finally, the catalytic mechanism and structural model that NHR recognizes 23S rRNA is proposed based on the structural and biochemical analyses. Thus, our systematic structural studies reveal the substrate recognition and modification by the nosiheptide-resistance methyltransferase.

Structural Basis for Cooperative Function of Mettl3 and Mettl14 Methyltransferases

“…The rest of the conserved cavity is likely for recognizing the RNA substrates. Similar to other MTDs, binding SAM or SAH does not induce a large conformational change of the domain, and most of the interactions are retained after the methyl transfer (Figure 3E) (Guja et al, 2013; Thomas et al, 2003; Yang et al, 2010). Nevertheless, when the apo structure is compared with the SAM or SAH bound states, we observe the largest movement in the loops that “fence” the perimeter of the cavity, especially near the carboxyl group of the bound cofactor (Figure S4C).…”

Binding Induced RNA Conformational Changes Control Substrate Recognition and Catalysis by the Thiostrepton Resistance Methyltransferase (Tsr)

“…This residue is absolutely conserved in the closely related nosiheptide resistance methyltransferase (Nhr; 23 S rRNA Am1067) and the avilamycin-resistance conferring methyltransferase AviRb (23 S rRNA Um2479), and functionally conserved in other SPOUT family members (8,37). The dramatic decrease in binding affinity is readily rationalized by examining the position of each Arg-162 residue in the dimeric Tsr structure.…”

“…The Escherichia coli 16 S and 23 S rRNAs contain 24 constitutively methylated nucleotides and the enzyme responsible for catalyzing each has been identified (6). Structural and biochemical studies of these enzymes and their homologs have provided significant insight into their interactions with the essential co-substrate S-adenosyl-L-methionine (AdoMet) 3 and their catalytic mechanisms (7,8). However, due to the relative paucity of structural studies methyltransferase-RNA substrate complexes, much less is understood about rRNA substrate recognition.…”

Functional roles inS‐adenosyl‐l‐methionine binding and catalysis for active site residues of the thiostrepton resistance methyltransferase

“…Organisms that produce thiostrepton ( Streptomycetes cyaneus , Streptomycetes laurentii )[10] express a SAM‐dependent methyltransferase, Tsr, that catalyzes a 2′‐ O ‐ribose methylation of an adenine nucleotide (A1067; Escherichia coli numbering) at the thiostrepton binding site[11], preventing its association and rendering the organisms resistant to its effects. Analogously, protection from the related thiopeptide nosiheptide, in the producing organism Streptomycetes actuosus , is afforded by a methyltransferase (Nhr) that shares 74% sequence similarity with Tsr[12].…”

“…Putative SPOUT methyltransferase genes have also been identified in the biosynthetic gene clusters of other thiopeptide‐producing bacteria, which may indicate that SPOUT‐enzyme RNA methylation is perhaps a more general form of thiopeptide resistance[22,23]. Structural studies show that SPOUT methyltransferases are functional homodimers, typified by an α/β Rossmann‐like fold with a deep trefoil knot at the C‐terminal end that binds SAM, and an active site near the dimeric interface that is constructed from residues contributed by both subunits[11,12,20,24–27]. Molecular models of RNA substrates bound to Tsr[11] or to other SPOUT methyltransferases[12,24,27] suggest that methyl transfer is accomplished by a single catalytic site, although these homodimeric enzymes appear capable of binding two SAM molecules.…”