The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate

Abstract

Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of ␤-lactam antibiotics is similar to that of the full-size membrane-bound PBP, the truncated PBP is expected to adopt the native folded conformation. The truncated PBP3 functions as a thiolesterase. In aqueous media and in the presence of millimolar concentrations of a properly structured amino compound, it catalyzes the aminolysis of the thiolester until completion, suggesting that the penicillinbinding module of PBP3 is designed to catalyze transpeptidation reactions. In contrast, the truncated PBP3 is devoid of glycan polymerization activity on the E. coli lipid II intermediate, suggesting that the non-penicillinbinding module of PBP3 is not a transglycosylase.The multimodular class B penicillin-binding protein 3 (PBP3) is a key element of the cell septation network in Escherichia coli (30). This tripartite protein consists of an M1-E56 membrane anchor-containing module that is fused to a G57-I237 non-penicillin-binding (n-PB) module that is fused to the D238-V577 PB module (7,12,16). The membrane anchor-free G57-V577 polypeptide chain of PBP3 which comprises the n-PB and PB modules has been overproduced in the periplasm of E. coli as an autonomous folding entity (9, 16). With "p" denoting "periplasmic," this truncated PBP3 is called PBP3p.Indirect experimental evidence suggests that the PB module of PBP3 is involved, one way or another, in peptide crosslinking during synthesis of the septal peptidoglycan (3,26). In contrast, the role of the n-PB module of PBP3 is a matter of controversy. According to Ishino and Matsuhashi (19), this module is a transglycosylase catalyzing glycan chain elongation from disaccharide (peptide) lipid II intermediates. According to van Heijenoort et al. (29), it is not.Because the thermostability and affinity for benzylpenicillin and cephalexin of the folded, membrane anchor-free polypeptide are unchanged in comparison with those of the membrane-bound PBP3, one can reasonably postulate that the conformation adopted by the truncated PBP3 reflects faithfully that of the native-state structure (9). ␤-Lactams and thiolester carbonyl donors and the disaccharide (peptide) lipid II intermediate were used to probe the enzymatic activities of the G57-V577 PBP3 and thus shed light on the possible functions of the protein in cell septation. The results are presented below.(Some of the work described in this paper is part of a dissertation presented by M.A. in partial fulfilment of a Ph.D. degree at the University of Liège.) MATERIALS AND METHODSPBP3p and carbonyl donor substrates. PBP3p was purified as described previously (9). It was stored frozen at Ϫ20°C in 10 mM Tris-HCl (pH 8.0)-10% (vol/vol) glycerol-10% (vol/vol) ethylene glycol-0.5 M NaCl. The esters and thiolesters were described previously (1, 2). [14 C]benzylpenicillin (54 Ci/mol) was from Amersham International (Buckinghamshire, United Kingdom). 5Ј-Fluoresceyl-glycyl-6-aminopenicillanate...

Probing the Catalytic Activity of a Cell Division-Specific Transpeptidase In Vivo with β-Lactams

“…Using ␤-lactams to probe the catalytic activity of PBP3 in growing cells makes it possible to assay the enzyme under conditions in which it is active in cell wall synthesis. These conditions cannot be replicated in vitro yet (1). We found that inhibition of cell division by a variety of methods had no detectable effect on the apparent rate of acylation of PBP3 with aztreonam or piperacillin but slightly reduced acylation with cephalexin.…”

“…Although PBP3 cannot be engaged in cell division under these conditions, it might still interact with some other division proteins. More recently, a truncated form of PBP3 that lacks its N-terminal membrane anchor sequence has been purified and shown to react with ␤-lactams at rates similar to those observed when native PBP3 is studied in isolated membranes (1). This finding argues against a requirement for allosteric activation, unless one supposes that the deletion itself activates the protein.…”

“…␤-Lactam antibiotics are often used as substrates to measure the catalytic activity of PBP3, because the physiological substrates used by the enzyme are not yet known (1). Some background will help clarify the principle of this assay.…”

The Transmembrane Helix of the Escherichia coli Division Protein FtsI Localizes to the Septal Ring

“…Interestingly, this peptide is a transmembrane helix (TMH). These findings help to clarify how targeting information is encoded in FtsI's primary sequence and demonstrate that a bacterial TMH can serve as a targeting signal.FtsI, also known as penicillin-binding protein 3 (PBP3), is a transpeptidase needed for cross-linking septal peptidoglycan (1,3,38). Previous studies from a number of laboratories have shown that FtsI is one of over a dozen proteins that localize to the division site, where they form a structure called the septal ring (for recent reviews, see references 12 and 43).…”

New Insights into the Final Stage of Bacterial Cell Division

“…The antibiotics chosen were the ß-lactams Aztreonam and Piperacillin. These compounds specifically inactivate PBP3 by acylating a serine of its transpeptidase catalytic site (Adam et al, 1997). For this analysis, MC4100 cells were grown in MS medium and at OD600nm=0.15, Aztreonam (0.25 µg/ml) was added to exponentially growing cells.…”