Hybrid proteins of the transglycosylase and the transpeptidase domains of PBP1B and PBP3 of Escherichia coli

Abstract

The construction of hybrid proteins of PBP1B and PBP3 has been described. One hybrid protein (PBP1B/3) contained the transglycosylase domain of PBP1B and the transpeptidase domain of PBP3. In the other hybrid protein, the putative transglycosylase domain of PBP3 was coupled to the transpeptidase domain of PBP1B (PBP3/1B). The hybrid proteins were localized in the cell envelope in a similar way as the wild-type PBP1B. In vitro isolates of the strains containing the hybrid proteins had a transglycosylase activity intermediate between that of wild-type PBP1B-producing strain and that of a PBP1B overproducer. Analysis with specific antibiotics against PBP1A/1B and PBP3 and mutant analysis in strains containing PBP3/1B revealed no detectable effects in vivo compared with wild-type strains. The same was shown for PBP1B/3 when the experiments were performed in a recA background. The data indicate that the hybrid proteins cannot replace native penicillinbinding proteins. This finding suggests that functional high-molecular-weight penicillin-binding protein specificity is at least in part determined by the unique combination of the two functional domains.The high-molecular-weight penicillin-binding proteins (HMW-PBPs) of Escherichia coli are responsible for the final stages of the synthesis of the peptidoglycan layer. E. coli contains four HMW-PBPs, called PBP1A, -1B, -2, and -3 (15). In vitro they have similar enzymatic activities, namely, a transglycosylase activity, situated in the amino-terminal part of the protein, and a transpeptidase activity, situated in its carboxyterminal part. If the HMW-PBPs are truly bifunctional, with the same biosynthetic activities, what is the basis for their functional differences in vivo? To explore which part, or combination of parts, of a specific HMW-PBP is responsible for its unique functional task, hybrid proteins might be helpful. For instance if the transpeptidase domain of one PBP is combined with the transglycosylase domain of another, does this new combination function, and if so, does it more resemble the PBP from which the transpeptidase part has been derived, or more the PBP from which the transglycosylase domain was derived?Construction of the fusion plasmids. To create hybrid proteins containing the transglycosylase domain of PBP1B and the transpeptidase domain of PBP3, and vice versa, the borders of the domains have to be localized. The parts that are important for the transpeptidase activity of the HMW-PBPs are all situated downstream of the active-site serine residue box of the respective HMW-PBPs (9). Little is known about the transglycosylase domain, its active center, and the importance of surrounding sequences in the HMW-PBPs.For PBP1B, the active-site serine of the transpeptidase domain is amino acid 510 (14). den Blaauwen (3) suggested that the transglycosylase domain of PBP1B resembles the active center of lysozyme and contains amino acid 304 (Asp). For the construction of the hybrid proteins, it is therefore assumed that a good border for PBP1B is about halfwa...

Penicillin-Binding Protein 2a of Streptococcus pneumoniae: Expression in Escherichia coli and Purification and Refolding of Inclusion Bodies into a Soluble and Enzymatically Active Enzyme

“…These observations may also suggest that the transglycosylase domain of class A PBPs, in particular, adopts a more hydrophobic folding conformation that allows the direct interaction of the transglycosylase domain of the protein with the membrane and its substrate. Consistent with this view, the transglycosylase of PBPs is known to utilize lipid intermediates of stem peptides as substrates (3,5,6,8,10,11) and is inhibited by moenomycin, a complex molecule with a long lipid tail (5,37). In contrast, the transpeptidase of PBPs appears to recognize only the stem peptide part of the lipid intermediates (1,2,4,23-25,27,32).…”

“…In contrast, the transglycosylase activity of class A high molecular weight PBPs is poorly characterized mainly because of the unavailability and complexity of its substrate, lipid intermediates of stem peptides (1)(2)(3)(4)(5)(6)8,10,11). The transglycosylase activity is routinely assayed by a complicated procedure involving the use of bacterial membrane preparations that contain radioisotope-labeled native substrates for the enzyme (3,6,8,10 -11).…”

Disruption of the gene encoding penicillin‐binding protein 2b (pbp2b) causes altered cell morphology and cease in exopolysaccharide production in Streptococcus thermophilus Sfi6

“…and belongs to the class B-type PBPs (Dowson et al, 1989). The C-terminal domain catalyses the transpeptidation of the interpeptide bridge and the N-terminal domain seems to be involved in the elongation of the glycan backbone but, as opposed to the N-terminal domain of class A PBPs, the transglycosylation function of the N-terminal domain of class B PBPs is still a matter of controversy (van Heijenoort et al, 1992;Ishino and Matsuhashi, 1981;Ghuysen, 1991;Zijderveld et al, 1995). The amino acid sequence of PBP 2b of S. thermophilus displayed the characteristics of class B PBPs proposed by Ghuysen (1991).…”

Inhibition of Transglycosylation Involved in Bacterial Peptidoglycan Synthesis

“…E. coli PBP3 is the most well studied class B protein, and some data are available on the role of the non-penicillin binding domains in forming membrane interactions and contributing to folding and stability [46,48]. Recently the transglycosylase domains of PBP1A and PBP3 were exchanged [49]. Although the hybrid proteins could not replace native PBP1A or PBP3 in vivo function, the hybrid containing the putative transglycosylase domain of PBP3 did appear to possess transglycosylase activity.…”