Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli

We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C beta-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acid of PBP 2 was identified as His633.     

Structural determinants required to target penicillin-binding protein 3 to the septum of Escherichia coli

In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.     

The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12

Penicillin-binding proteins 1A and 1B of Escherichia coli are the major peptidoglycan transglycosylase-transpeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93 500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1B, was also determined. Penicillin-binding protein 1B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1A and 1B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identity. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C beta-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1A and 1B and the other penicillin-binding proteins and beta-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1A, 1B and 3, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The other region was 19-40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.     

Construction of a mutant of Escherichia coli that has deletions of both the penicillin-binding protein 5 and 6 genes

A mutant of Escherichia coli has been constructed with deletions of the genes encoding penicillin-binding protein 5 (dacA) and penicillin-binding protein 6 (dacC). The construction of this mutant establishes that the complete loss of the two most abundant species of penicillin-binding proteins can be tolerated by E. coli. Moreover, the double deletion mutant had the same growth rate and morphology as an isogenic dacA+ dacC+ strain.     

Deletion of the penicillin-binding protein 5 gene of Escherichia coli.

A strain of Escherichia coli that has a deletion of the entire dacA gene has been constructed. The complete lack of penicillin-binding protein 5 in this strain establishes that the activity of this protein is not essential for the growth of E. coli.     

Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.     

Membrane topology of penicillin-binding protein 3 of Escherichia coli

The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.     

A mutant of Escherichia coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase IA.

A mutant of Escherichia coli defective in penicillin-binding protein 5 activity was isolated. The mutation (pfv) was shown to be located at 14.0 min on the E. coli chromosome map. Loss of penicillin-binding protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to beta-lactam antibiotics. We conclude that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity and that the enzyme activity, in vivo, protects E. coli cells from killing by low inhibitory concentrations of beta-lactam antibiotics.     

Lysis of Escherichia coli by beta-lactam antibiotics: deletion analysis of the role of penicillin-binding proteins 1A and 1B

Deletions of the ponA and ponB genes of Escherichia coli have been constructed in vitro and recombined into the chromosome to produce strains that completely lack penicillin-binding protein 1A or penicillin-binding protein 1B. In each case a DNA fragment internal to the gene was replaced by a fragment encoding an antibiotic resistance. The ponA and ponB deletions can therefore be readily introduced into other E. coli strains by P1 transduction of the antibiotic resistance. Although the complete absence of penicillin-binding protein 1A or penicillin-binding protein 1B was tolerated, the absence of both of these proteins was shown to result in bacterial lysis.     

Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (greater than 1000-fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin-binding protein 2B from three penicillin-sensitive strains of S. pneumoniae and from a penicillin-resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin-sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin-resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the 'penicillin-resistant' form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin-binding site.     

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.     

Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin-binding protein 2 of Escherichia coli K12

We have determined the nucleotide sequence of the pbpA gene encoding penicillin-binding protein (PBP) 2 of Escherichia coli. The coding region for PBP 2 was 1899 base pairs in length and was preceded by a possible promoter sequence and two open reading frames. The primary structure of PBP 2, deduced from the nucleotide sequence, comprised 633 amino acid residues. The relative molecular mass was calculated to be 70867. The deduced sequence agreed with the NH2-terminal sequence of PBP 2 purified from membranes, suggesting that PBP 2 has no signal peptide. The hydropathy profile suggested that the NH2-terminal hydrophobic region (a stretch of 25 non-ionic amino acids) may anchor PBP 2 in the cytoplasmic membrane as an ectoprotein. There were nine homologous segments in the amino acid sequence of PBP 2 when compared with PBP 3 of E. coli. The active-site serine residue of PBP 2 was predicted to be Ser-330. Around this putative active-site serine residue was found the conserved sequence of Ser-Xaa-Xaa-Lys, which has been identified in all of the other E. coli PBPs so far studied (PBPs 1A, 1B, 3, 5 and 6) and class A and class C beta-lactamases. In the higher-molecular-mass PBPs 1A, 1B, 2 and 3, Ser-Xaa-Xaa-Lys-Pro was conserved. In the putative peptidoglycan transpeptidase domain there were six amino acid residues, which are common only in the PBPs of higher molecular mass.     

Crystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams.     

Mode of membrane insertion and sequence of a 32-amino acid peptide stretch of the penicillin-binding protein 4 of Enterococcus hirae

Analysis of water-soluble derivatives of the Enterococcus hirae 75-kDa membrane-bound penicillin-binding protein 4 (PBP4) has yielded the amino acid sequence of a 32-amino acid polypeptide stretch. This peptide is similar to peptide segments known to occur in the N-terminal domain of high-Mr PBPs of class B. The E. hirae PBP4 probably belongs to the same class. It is anchored in the membrane at the N-terminus of the polypeptide chain.     

Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli

The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the [14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.     

Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin

The location of amino acid substitutions that allow an enzyme to discriminate between the binding of its normal substrate and a substrate analogue may be used to identify regions of the polypeptide that fold to form the substrate binding site. We have isolated a large number of cephalexin-resistant mutants of Escherichia coli in which the resistance is due to the production of altered forms of penicillin-binding protein 3 that have reduced affinity for the antibiotic. Using three mutagens, and a variety of selection procedures, we obtained only five classes of mutants which could be distinguished by their patterns of cross-resistance to other beta-lactam antibiotics. The three classes of mutants that showed the highest levels of resistance to cephalexin were cross-resistant to several other cephalosporins but not to penicillins or to the monobactam, aztreonam. The penicillin-binding protein 3 gene from 46 independent mutants was cloned and sequenced. Each member of the five classes of cephalexin-resistant mutants had the same amino acid substitution in penicillin-binding protein 3. The mutants that showed the highest levels of resistance to cephalexin had alterations of either Thr-308 to Pro, Val-344 to Gly, or Asn-361 to Ser. The Thr-308 to Pro substitution had occurred within the beta-lactam-binding site since the adjacent residue (Ser-307) has been shown to be acylated by benzylpenicillin. The Asn-361 to Ser change occurred in a region that showed substantial similarity to regions in both penicillin-binding protein 1A and 1B and may also define a residue that is located within the beta-lactam-binding site in the three-dimensional structure of the enzyme.     

Reduced heat resistance of mutant spores after cloning and mutagenesis of the Bacillus subtilis gene encoding penicillin-binding protein 5.

Part of the gene encoding penicillin-binding protein 5 from Bacillus subtilis 168 was cloned in Escherichia coli with a synthetic oligonucleotide as a hybridization probe. The gene was designated dacA by analogy with E. coli. The nucleotide sequence was determined, and the predicted molecular mass was 45,594 daltons (412 amino acids). A comparison of the predicted amino acid sequence with that of the E. coli penicillin-binding protein 5 indicated that these enzymes showed about 25% identity. The B. subtilis dacA gene was mutated by integration of a plasmid into the structural gene by homologous recombination. A comparison of the mutant and control strains revealed that (i) the mutant lacked detectable penicillin-binding protein 5, (ii) the D-alanine carboxypeptidase activity of membranes isolated from the mutant was only 5% of that measured in membranes from the control strain, (iii) the mutant cells showed apparently normal morphology only during exponential growth, and after the end of exponential phase the cells became progressively shorter, (iv) the mutant sporulated normally except that the forespore occupied about two-thirds of the mother cell cytoplasm and, during its development, migrated towards the center of the mother cell, and (v) purified mutant spores were 10-fold less heat resistant but possessed normal refractility and morphology. Preliminary chemical analysis indicated that the structure of the cortex of the mutant was different.     

Trapping of the substrate-derived acyl enzyme intermediate of purified penicillin-binding protein 1a of Escherichia coli

Purified penicillin-binding protein 1a of Escherichia coli formed an acyl enzyme intermediate with the highly reactive synthetic substrate diacetyl-L-lysyl-D-alanyl-D-lactate at acid pH, although in extremely low yields.     

Activity of penicillin-binding protein 3 from Escherichia coli.

The activity of penicillin-binding protein 3 of Escherichia coli has been studied both in vivo and in ether-permeabilized cells. The peptidoglycan transpeptidase activity of penicillin-binding protein 3 appears to use either nascent or exogenously added UDP-N-acetylmuramyl tripeptide-derived substrates as acceptors. By means of a defilamentation system which elicited the activity of penicillin-binding protein 3 in vivo, the structure of peptidoglycan made by this enzyme has been elucidated. This peptidoglycan, very probably of septal location, contained increased amounts of cross-linked peptidoglycan as well as a higher ratio of tripeptide-containing cross-linked subunits.