The glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae catalyzes the polymerization of murein glycan chains

The bacterial peptidoglycan consists of glycan chains of repeating beta-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The beta-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K(m) and k(cat)/K(m) were measured at 40.6 micro M (+/- 15.5) and 1 x 10(-3) M(-1) s(-1), respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.     

Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.     

Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233.     

Identification,Purification, and Characterization of Transpeptidase and Glycosyltransferase Domains of Streptococcus pneumoniae Penicillin-Binding Protein 1a

Resistance to β-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both β-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.The synthesis of the bacterial cell wall requires cytoplasmic and periplasmic enzymes. The final steps of peptidoglycan biosynthesis occur outside the cytoplasmic membrane, and they are catalyzed by membrane-bound penicillin-binding proteins (PBPs). PBPs play essential roles in cell division and morphology (6, 20, 31). Based upon their molecular sizes and amino acid sequence similarities, PBPs can be classified into two groups (6): low-molecular-weight (low-M_r) PBPs, which act as d,d-carboxypeptidases, and high-molecular-weight (high-M_r) PBPs, which carry transpeptidase (TP) and glycosyltransferase (GT) activities. The high-M_r group can be further divided into bifunctional enzymes with TP and GT activities (class A) and monofunctional TP enzymes (class B).β-Lactam antibiotics bind with high affinity specifically to d,d-carboxypeptidase and TP domains because of their structural similarity to the natural substrates, the stem peptides. This binding results in the formation of a covalent acyl-PBP enzyme complex, leading to the inactivation of PBPs.High-M_r PBPs are multidomain proteins (6). The three-dimensional structure of Streptococcus pneumoniae PBP 2x (class B high-M_r PBP) illustrates this domain organization (25). The only non-penicillin-binding domain of known function is the GT domain, corresponding to the N-terminal region of class A PBPs. This GT activity, clearly identified in Escherichia coli PBP 1b, is difficult to measure (23, 29, 31–35). It is insensitive to penicillin but sensitive to moenomycin, an antibiotic which is not used for human therapy (23, 29, 32, 33).S. pneumoniae is one of the major human pathogens of the upper respiratory tract, causing pneumonia, meningitis, and ear infections. Six PBPs have been identified in S. pneumoniae: high-M_r PBPs 1a, 1b, 2a, 2x, and 2b and low-M_r PBP 3 (8). PBPs 1a, 1b, and 2a belong to class A, while PBPs 2x and 2b are monofunctional class B proteins. Deletion of pbp2x and pbp2b in S. pneumoniae is lethal for the bacteria, while the deletion of pbp1a is tolerated (11), probably due to compensation by PBP 1b. This has been observed for E. coli class A PBP 1a, whose deletion can be compensated for by PBP 1b (36). In clinical isolates of resistant pneumococci, pbp1a, pbp2x, and pbp2b genes were shown to present a mosaic organization, encoding PBPs with reduced affinity for β-lactam antibiotics (2, 5, 15, 18). The specific resistance to ceftriaxone and cefotaxime of S. pneumoniae from the hospital environment is mediated by modification of PBP 2x and PBP 1a (22). Furthermore, gene transfer of pbp1a, pbp2x, and pbp2b from resistant strains conferred penicillin resistance on sensitive S. pneumoniae strains under laboratory conditions (2–4, 14, 15, 27, 30).The effort to overcome resistance to antibiotics in S. pneumoniae might therefore benefit from a detailed understanding of the molecular basis of TP and GT activities. The GT domain represents a new potential target for novel nonpenicillin antibiotics. Here, we delineate the GT and TP domains of S. pneumoniae PBP 1a* (a water-soluble form of PBP 1a) by limited proteolytic digestion and expression of recombinant domains. The TP activity of PBP 1a* and that of the isolated TP domain were compared. We also present evidence for an interaction between the isolated GT domain and moenomycin.     

Microplate assay for inhibitors of the transpeptidase activity of PBP1b of Escherichia coli

The transpeptidase (TP) activity of penicillin-binding proteins (PBPs), target of the beta-lactam antibiotics, is a well-validated antibacterial drug target. The TP activity of PBP1b converts un-cross-linked peptidoglycan to the cross-linked form. Directly measuring TP activity is difficult because cross-linked and un-cross-linked peptidoglycan have very similar chromatographic properties. The authors report a microdilution plate method to directly measure the TP enzyme activity, uncoupled from the transglycosylase (TG), for detection of TP inhibitors. Escherichia coli membranes were incubated with 100 mM ampicillin, followed by removal of unbound ampicillin. The substrate for the TP, un-cross-linked peptidoglycan, was prepared by incubating these membranes with peptidoglycan sugar precursors, 1 of which was radiolabeled. Subsequently, solubilized PBP1b was added and TP activity assayed. The cross-linked peptidoglycan formed was monitored by addition of wheat germ agglutinin scintillation proximity assay beads plus N-laurylsarcosine, which selectively captures cross-linked peptidoglycan. The PBP1bcatalyzed activity was inhibited by penicillin G but not by cephalexin or cephradine, which have higher affinity for PBP1a. Moenomycin, a TG inhibitor, also inhibited TP activity. Because this is a true enzyme assay, it has the potential to detect novel, non-beta-lactam TP inhibitors and could lead to the discovery of new antibacterial agents.     

Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3.

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.     

Kinetic characterization of the monofunctional glycosyltransferase from Staphylococcus aureus

The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate-N-acetylglucosaminyl-N-acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the Staphylococcus aureus MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of approximately 5,800 M(-1) s(-1) and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn2+. The properties of S. aureus MGT are distinct from those of S. aureus PBP2 and Escherichia coli MGT, but they are similar to those of E. coli PBP1b. We examined the role of the conserved Glu100 of S. aureus MGT (equivalent to the proposed catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in S. aureus MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site.     

Fluctuations in nuclear envelope's potential mediate synchronization of early neural activity

Penicillin binding proteins (PBPs) catalyze essential steps in the biosynthesis of peptidoglycan, the main component of the bacterial cell wall. PBPs can harbor two catalytic domains, namely the glycosyltransferase (GT) and transpeptidase (TP) activities, the latter being the target for β-lactam antibiotics. Despite the availability of structural information regarding bi-functional PBPs, little is known regarding the interaction and flexibility between the TP and GT domains. Here, we describe the structural characterization in solution by small angle X-ray scattering (SAXS) of PBP1b, a bi-functional PBP from Streptococcus pneumoniae. The molecule is present in solution as an elongated monomer. Refinement of internal coordinates starting from a homology model yields models in which the two domains are in an extended conformation without any mutual contact compatible with the existence of restricted mobility.     

Two distinct transpeptidation reactions during murein synthesis in Escherichia coli.

Murein synthesized in ether-permeabilized cells of Escherichia coli deficient in individual penicillin-binding proteins (PBPs) and in the presence of certain beta-lactam antibiotics was analyzed by high-pressure liquid chromatography separation of the muramidase split products. PBP 1b was found to to be the major murein synthesizing activity that was poorly compensated for by PBP 1a. A PBP 2 mutant as well as mecillinam-inhibited cells showed increased activity in the formation of oligomeric muropeptides as well as UDP-muramylpeptidyl-linked muropeptides, the reaction products of transpeptidation, bypassing the lipid intermediate. In contrast, penicillin G and furazlocillin severely inhibited these reactions but stimulated normal dimer production. It is concluded that two distinct transpeptidases exist in E. coli: one, highly sensitive to penicillin G and furazlocillin, catalyzes the formation of hyper-cross-linked muropeptides, and a second one, quite resistant to these antibiotics, synthesizes muropeptide dimers.     

Interaction of monoclonal antibodies with the enzymatic domains of penicillin-binding protein 1b of Escherichia coli.

Monoclonal antibodies (MAbs) against four different antigenic determinants of penicillin-binding protein (PBP) 1b were used to study the transglycosylase and transpeptidase activities of PBP 1b. Enzyme kinetics in the presence of and without the MAbs were determined, and the synthesized murein was analyzed. Two MAbs against the transglycosylase domain of PBP 1b appeared to inhibit this reaction. One MAb inhibited only the transpeptidase reaction, and one inhibited both enzymatic activities of PBP 1b. The latter two MAbs bound to the transpeptidase domain of PBP 1b. The following major conclusions were deduced from the results. (i) Transpeptidation is the rate-limiting step of the reaction cascade, and it is dependent on the product of transglycosylation. (ii) PBP 1b has only one type of transpeptidase activity, i.e., a penta-tetra transpeptidase activity. (iii) PBP 1b is probably a globular protein which has two intimately associated enzymatic domains.     

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.     

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli

The penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported N-acetyl glucosaminyl-beta-1, 4-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl+ ++- (L)-D-alanyl-D-alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46-N844) PBP1bgamma. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bgamma possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an congruent with 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of congruent with 39 000 M-1 s-1. Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 gamma-COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4-OH group of a nucleophile N-acetylglucosamine. Asp-234 of motif 1 or Glu-290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4-OH group of the N-acetylglucosamine. In turn, Tyr-310 of motif 4 is an important component of the amino acid sequence-folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447-N844 acyl transferase module, which possesses the catalytic centre-defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active-site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser-510-assisted double-proton shuttle are glycan strands substituted by cross-linked tetrapeptide-pentapeptide and tetrapeptide-tetrapeptide dimers and uncross-linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on D-alanyl-D-alanine-terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross-linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.     

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.     

In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli

The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric triand tetrapeptides in the sacculi were the acceptors.     

Structural analysis of an "open" form of PBP1B from Streptococcus pneumoniae

The class A PBP1b from Streptococcus pneumoniae is responsible for glycosyltransferase and transpeptidase (TP) reactions, forming the peptidoglycan of the bacterial cell wall. The enzyme has been produced in a stable, soluble form and undergoes time-dependent proteolysis to leave an intact TP domain. Crystals of this TP domain were obtained, diffracting to 2.2 A resolution, and the structure was solved by using molecular replacement. Analysis of the structure revealed an "open" active site, with important conformational differences to the previously determined "closed" apoenzyme. The active-site nucleophile, Ser460, is in an orientation that allows for acylation by beta-lactams. Consistent with the productive conformation of the conserved active-site catalytic residues, adjacent loops show only minor deviation from those of known acyl-enzyme structures. These findings are discussed in the context of enzyme functionality and the possible conformational sampling of PBP1b between active and inactive states.     

Topographical and functional investigation of Escherichia coli penicillin-binding protein 1b by alanine stretch scanning mutagenesis.

Penicillin-binding proteins (PBPs) are the targets of beta-lactam antibiotics. We have used a systematic five-alanine substitution method (called ASS [alanine stretch scanning] mutagenesis) to investigate the functional or structural role of various stretches of amino acids in the PBP1b of Escherichia coli. To probe the specific activity of each variant, the antibiotic discs assay was used with strain QCB1 (delta ponB) in the presence of cefaloridine, which totally inhibits the complementing action of PBP1a. This in vivo test has been combined with a quick and efficient in vitro test of the penicillin-binding activity of each of these variants with fluorescent penicillin. This approach has enabled us to show an unexpected role of the N-terminal and C-terminal tails of PBP1b. Moreover, we have established the correct position in PBP1b of the SMN motif that, with the SXXK and the KTG motifs, constitutes the signature of the penicilloyl serine transferases family. Finally, we have shown that the transglycosylase and the transpeptidase domains are separated by an inert linker region, where substitutions and insertions can be made without hindering the in vivo and in vitro activity of the protein.     

Mutational analysis of the Streptococcus pneumoniae bimodular class A penicillin-binding proteins.

One group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding-transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the three Streptococcus pneumoniae class A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disrupted pbp2a gene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors in S. pneumoniae.     

Regulation of Ca2+/calmodulin-dependent protein kinase II by brain gangliosides

Purified rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 microM. Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 microM) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis. CaMK 281-309 strongly inhibited kinase activity (IC50 = 0.2 microM). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

Synthesis of mono- and disaccharide analogs of moenomycin and lipid II for inhibition of transglycosylase activity of penicillin-binding protein 1b

Three types of mono- and disaccharides 3a,b, 4a–c, 5, and some chaetomellic acid A analogs 6 and 42–44 were synthesized as potential inhibitors of the transglycosylase activity of penicillin-binding protein 1b (PBP1b), a key bacterial enzyme responsible for the formation of the polysaccharide backbone of peptidoglycan as well as for cross-linking of its peptide portions. The target compounds combine structural features of both the active portion of moenomycin and the natural PBP1b substrate, lipid II. The desired skeletons were obtained in a convergent fashion involving attachment of the lipid-alkylated glyceric acid moieties 11a,b to the corresponding carbohydrate-containing phosphonic acids 23, 24a, and 24b. Compounds 3a,b were prepared to verify the distance requirements between the sugar and the noncleavable C-phosphonate moieties. Compounds 4a–c were synthesized to examine the importance of the first sugar unit of moenomycin, a known inhibitor of transglycosylase catalysis by PBP1b, with respect to antibiotic activity. These were prepared by condensation of 11a,b with 28a and 28c, which were made by glycosylation of 3-bromopropanol with oxazolines 25a,b, and Arbuzov reaction with triethyl or trimethyl phosphite, followed by dealkylation with bromotrimethylsilane. Compound 5 was generated to verify the possibility of using a dicarboxylate group to mimic the diphosphate of lipid II. It was synthesized by coupling of alcohol 31 with -trichloroacetimidate 34. Chaetomellic acid A analogs were prepared by a Michael addition to dimethyl acetylenedicarboxylate. With the exception of 3b, all of the target compounds were found to inhibit PBP1b, albeit with modest potency.     

Crystal structure of penicillin-binding protein 1a (PBP1a) reveals a mutational hotspot implicated in beta-lactam resistance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen whose infections have been treated with beta-lactam antibiotics for over 60 years, but the proliferation of strains that are highly resistant to such drugs is a problem of worldwide concern. Beta-lactams target penicillin-binding proteins (PBPs), membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. Bifunctional PBPs catalyze both the polymerization of glycan chains (glycosyltransfer) and the cross-linking of adjacent pentapeptides (transpeptidation), while monofunctional enzymes catalyze only the latter reaction. Although S. pneumoniae has six PBPs, only three (PBP1a, PBP2x, PBP2b) are major resistance determinants, with PBP1a being the only bifunctional enzyme. PBP1a plays a key role in septum formation during the cell division cycle and its modification is essential for the development of high-level resistance to penicillins and cephalosporins. The crystal structure of a soluble form of pneumococcal PBP1a (PBP1a*) has been solved to 2.6A and reveals that it folds into three domains. The N terminus contains a peptide from the glycosyltransfer domain bound to an interdomain linker region, followed by a central, transpeptidase domain, and a small C-terminal unit. An analysis of PBP1a sequences from drug-resistant clinical strains in light of the structure reveals the existence of a mutational hotspot at the entrance of the catalytic cleft that leads to the modification of the polarity and accessibility of the mutated PBP1a active site. The presence of this hotspot in all variants sequenced to date is of key relevance for the development of novel antibiotherapies for the treatment of beta-lactam-resistant pneumococcal strains.