Abstract
It is largely accepted that serine beta-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with beta-lactam antibiotics, such as penicillins. On the other hand, beta-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed. Besides, all known PBPs and beta-lactamases share very low sequence similarities, thus rendering it difficult to understand how a PBP could evolve into a beta-lactamase. In this study, we identified a new family of cyanobacterial PBPs featuring the highest sequence similarity with the most widespread class A beta-lactamases. Interestingly, the Omega-loop, which, in the beta-lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue. From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Penicillin degradation pathways as well as acylation and deacylation rates are characteristic of PBPs. In a first attempt to generate beta-lactamase activity, a 90-fold increase in deacylation rate was obtained by introducing a glutamate in the shorter Omega-loop.
Highlights
D-Alanyl-D-alanine peptidases are enzymes involved in the synthesis of the peptidoglycan, the bacterial cell wall constituent that is responsible for the cell shape and resistance to osmotic pressure [1]
Primary Sequence Analysis of penicillin-binding proteins (PBPs)-A Family—By searching bacterial genomes for genes encoding penicillin recognizing proteins, we identified a whole family of cyanobacterial proteins annotated as putative class A -lactamases but with a 6-amino acids deletion in the region of the ⍀-loop and missing the essential Glu166
Physiological Role of PBP-A—We have shown that PBP-A is a penicillin-binding protein that hydrolyzes -lactam substrates more than 6 orders of magnitude more slowly than TEM-1 -lactamase
Summary
DD-peptidases are called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with -lactam antibiotics, such as penicillins. The ⍀-loop, which, in the -lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Sequence alignments and comparative analysis of the structures and the reaction mechanisms indicate that these enzymes are phylogenetically linked [3, 16, 17] and that each class of -lactamase evolved most probably from an ancestral PBP, bringing to the active site an efficient catalysis mechanism for the deacylation reaction. A New Family of PBPs mutation L158E in the shorter ⍀-loop afforded a 90-fold improved deacylation of the penicilloyl-enzyme
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