Protein-engineering of the low affinity Penicillin Binding Protein 5 that confers penicillin resistance to Enterococcus hirae

MOLLERACH M; COYETTE J

Leuven, Bélgica

Simposio; Symposium of the National Comitee of Microbiology; 1994

National Comitee of Microbiology.- Belgium

The relatively low sensitivity of Enterococcus hirae ATCC 9790 to penicillin was associated with the presence of a high molecular weight penicillin binding protein (PBP) of low affinity, the 71 kDa PBP5. This PBP and the low affinity Staphylococcus aureus PBP2' appeared to have an additional 110 aminoacid polypeptide inserted between the membrane anchor and the N-terminal domain found in other class B high molecular weight PBPs. These polypeptide insertions seemed to have specific predicted secondary structures. Different deletions inside the 110 aminoacid module were produced using the nested deletion procedure after BalI digestion and preserving the reading frame. Four truncated pbp5 genes were obtained. They were sequenced and expressed in Escherichia coli. Western blot analysis confirmed the production of shortened PBP5s. Penicillin binding assays with benzyl- 14C-penicillin showed that they had lost their binding activity. The presence of the deleted fragments seemed to be essential for the penicillin binding activity in the native protein. However that inactivity could be due to the incorrect folding of the truncated PBPs as it was previously demonstrated that a 44 kDa tryptic fragment, isolated from membrane bound PBP5 and equivalent to the C-terminal domain was able to react with 14C-penicillin exactly as the native PBP5.