PBP-A, a cyanobacterial DD-peptidase with high specificity for amidated muropeptides, imposes a pH-dependent fitness cost in Escherichia coli as a consequence of promiscuous activity

Abstract

Abstract Penicillin binding proteins (PBPs) are involved in biosynthesis, remodeling and recycling of peptidoglycan (PG) in bacteria. PBP-A from Thermosynechococcus elongatus belongs to a cyanobacterial family of enzymes sharing close structural and phylogenetic proximity to class A beta-lactamases. With the aim of converting PBP-A into a beta-lactamase, we expressed the enzyme in the periplasm of Escherichia coli but failed in directed evolution experiments and observed growth defect associated with the enzyme activity. To further explore the molecular origins of the fitness cost, we decided to characterize deeper the activity of PBP-A both in vitro and in vivo. We found that PBP-A is an enzyme endowed with DD-carboxypeptidase and DD-endopeptidase activities, featuring high specificity towards muropeptides amidated on the D-iso-glutamyl residue. We also show that its promiscuous activity on non-amidated peptidoglycan deteriorates E. coli’s envelope and generates the fitness cost, which is much higher under acidic conditions where substrate discrimination is mitigated. Besides expanding our knowledge on the biochemical activity of PBP-A, this work also highlights how promiscuity may hinder rather than promote enzyme evolution in nature or in the laboratory.