Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm

Abstract

The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K(D) (binding constant) of 17.6 microM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.

FIG. 1.

FmrO MT family sequence analysis, amino acid conservation, and homology modeling. (A) Phylogenetic tree of MTs from aminoglycoside-producing actinomycete strains. Numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analyses of 1,000 resampled data sets (27). The scale bar represents 0.1 amino acid substitutions per position. (B) LOGOS representation (6, 29) of the FmrO MT family sequence alignment used to construct the phylogenetic tree in panel A. Asterisks denote the conserved, putative key residues for cofactor binding (red), the catalysis of methyl group transfer (blue), and target site recognition (green). The position of one additional mutation (N179A) made as a control in the analysis of SAM binding is marked with an arrow. (C) ConSurf model of the Sgm protein with the clusters of key residues identified in panel B highlighted. The color code bar above the model indicates amino acid conservation from variable residues (light blue) to highly conserved residues (purple). (D) Cartoon representation of the Sgm homology model with amino acids targeted for site-directed mutagenesis within the proposed SAM binding pocket (red) and putative catalytic residues (blue) shown in stick representation. Additional amino acid changes (discussed in the text) are the mutation at a variable site near the SAM pocket (N179A; magenta) and two spontaneous mutations identified during selection (cyan). The figure was prepared using PyMOL (http://www.pymol.org).

FIG. 2.

CD spectroscopy analysis of Sgm. CD spectrum of purified wild-type Sgm collected at 20°C and used for analysis of protein secondary structure content via Dichroweb (Table 2). deg., degrees.

FIG. 3.

In vitro methylation activities of wild-type and mutant Sgm proteins. (A) Comparison of wild-type Sgm methylation activity against sensitive 30S (S-30S; solid line) and resistant 30S (R-30S; dotted line) ribosomal subunit substrates. MT-free cell extract from E. coli lacking an Sgm-encoding plasmid was used as a negative control (EC cell extract; dashed line) in all methylation assays and indicates background levels of methylation activity. (B) Methylation activities against sensitive 30S ribosomal subunits of Sgm proteins mutated at a nonconserved residue near the SAM pocket (N179A; squares) and at putative key residues for cofactor binding, as follows: G135A (circles), D156A (triangles), and D182A (diamonds). (C) Methylation activities against sensitive 30S ribosomal subunits of Sgm proteins mutated at putative key residues for methyl group transfer, as follows: K199A (circles), E205A (triangles), R236A (squares), and E267A (diamonds). In panels B and C, the wild-type (solid line) and negative control (dashed line) data are the same as in panel A.

FIG. 4.

Isothermal titration calorimetric analysis of SAM binding to Sgm. (A) Titration of SAM (via syringe) into the wild-type Sgm protein (sample cell). The upper panel shows the baseline-corrected titration data, and the lower panel shows the binding isotherm fit to a model for a single SAM binding site. Equivalent titration data are shown for N179A (B), D182A (C), and R236A (D) mutant Sgm proteins.