A natural polymorphism in beta-lactamase is a global suppressor

Abstract

A M182T substitution was discovered as a second-site suppressor of a missense mutation in TEM-1 beta-lactamase. The combination of the M182T substitution with other substitutions in the enzyme indicates the M182T substitution is a global suppressor of missense mutations in beta-lactamase. The M182T substitution also is found in natural variants of TEM-1 beta-lactamase with altered substrate specificity that have evolved in response to antibiotic therapy. The M182T substitution may have been selected in natural isolates as a suppressor of folding or stability defects resulting from mutations associated with drug resistance. This pathway of protein evolution may occur in other targets of antimicrobial drugs such as the HIV protease.

Figure 1

Steady-state levels of wild-type and mutant β-lactamases evaluated by immunoblotting. (A) Immunoblot of β-lactamase mutants in E. coli XL1-Blue. Positions are: 1, wild-type TEM-1 β-lactamase; 2, L76N; 3, L76S; 4, M182T; 5, L76N:M182T; 6, L76S:M182T; 7, M69I; 8, M69I:M182T; 9, I47Y:E48C; 10, I47Y:E48C:M182T. (B) Immunoblot of β-lactamase mutants in E. coli SB646. The order of mutants is as in A.

Figure 2

Diagram of the TEM-1 β-lactamase structure (20) showing the positions of residues I47 and E48 (red), M69 (yellow), L76 (magenta), and M182 (cyan). The position of the active site is indicated by the ball-and-stick representation of the catalytic residues S70, S130, and E166. Figure was made using molscript (21).

Figure 3

Graph of specific activities of β-lactamase mutants at increasing temperatures. The x axis displays the temperature at which reactions were performed while the y axis is the specific activity that was measured as mM ampicillin hydrolyzed per mg of total protein per min. □, wild-type TEM-1 β-lactamase; ▪, M69I; ○, M69I:M182T; •, M182T.