Probing the active site of beta-lactamase R-TEM1 by informational suppression

Using a new extended set of 13 amber suppressors in E coli, systematic amino-acid replacements were performed at positions 104(E) and 238(G) of TEM-1 beta-lactamase from PUC19. The enzyme is tolerant to most substitutions tested at position 104. Missense revertants E104K, E104S or E104Y exhibited only minor changes in enzyme activity with respect to wild-type TEM-1. Several substitutions at position 238 resulted in a new cefotaxime hydrolysing capacity, but to an extent that did not confer cefotaxime resistance for the bacteria producing the mutated enzymes. Only when the mutations at codons 104 and 238 were combined on the same gene, did a true cefotaxime resistant phenotype appear, mimicking the situation encountered with 3rd generation cephalosporins resistant clinical isolates.