Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.     

Sequences near the active site in chimeric penicillin binding proteins 5 and 6 affect uniform morphology of Escherichia coli

Penicillin binding protein (PBP) 5, a DD-carboxypeptidase that removes the terminal D-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp(218) and Lys(219) residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5.     

Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli

Escherichia coli has 12 recognized penicillin binding proteins (PBPs), four of which (PBPs 4, 5, and 6 and DacD) have DD-carboxypeptidase activity. Although the enzymology of the DD-carboxypeptidases has been studied extensively, the in vivo functions of these proteins are poorly understood. To explain why E. coli maintains four independent loci encoding enzymes of considerable sequence identity and comparable in vitro activity, it has been proposed that the DD-carboxypeptidases may substitute for one another in vivo. We tested the validity of this equivalent substitution hypothesis by investigating the effects of these proteins on the aberrant morphology of DeltadacA mutants, which produce no PBP 5. Although cloned PBP 5 complemented the morphological phenotype of a DeltadacA mutant lacking a total of seven PBPs, controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper functioning of PBP 5. By examining a set of mutants from which multiple PBP genes were deleted, we found that significant morphological aberrations required the absence of at least three different PBPs. The greatest defects were observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endopeptidases (either PBP 4 or PBP 7). The results further differentiate the roles of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recently determined crystal structure of PBP 5, suggest possible mechanisms by which these PBPs may contribute to maintenance of a uniform cell shape in E. coli.     

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.     

Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.     

Properties and crystallization of a genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12

Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.     

Crystal structure of penicillin binding protein 4 (dacB) from Escherichia coli, both in the native form and covalently linked to various antibiotics

The crystal structure of penicillin binding protein 4 (PBP4) from Escherichia coli, which has both DD-endopeptidase and DD-carboxypeptidase activity, is presented. PBP4 is one of 12 penicillin binding proteins in E. coli involved in the synthesis and maintenance of the cell wall. The model contains a penicillin binding domain similar to known structures, but includes a large insertion which folds into domains with unique folds. The structures of the protein covalently attached to five different antibiotics presented here show the active site residues are unmoved compared to the apoprotein, but nearby surface loops and helices are displaced in some cases. The altered geometry of conserved active site residues compared with those of other PBPs suggests a possible cause for the slow deacylation rate of PBP4.     

The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli

The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Substitution of Alanine at Position 184 with Glutamic Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis> PBP5 Ω-Like Loop Introduces a Moderate Cephalosporinase Activity

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic β-lactam resistance. Though PBP5 does not have β-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A β-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in β-lactamases. It is speculated that β-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5_A184E. Expression of PBP5_A184E in E. coli ?PBP5 mutant elevates the β-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5_A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and β-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.     

Cloning and characterization of PBP 1C, a third member of the multimodular class A penicillin-binding proteins of Escherichia coli.

All proteins of Escherichia coli that covalently bind penicillin have been cloned except for the penicillin-binding protein (PBP) 1C. For a detailed understanding of the mode of action of beta-lactam antibiotics, cloning of the gene encoding PBP1C was of major importance. Therefore, the structural gene was identified in the E. coli genomic lambda library of Kohara and subcloned, and PBP1C was characterized biochemically. PBP1C is a close homologue to the bifunctional transpeptidases/transglycosylases PBP1A and PBP1B and likewise shows murein polymerizing activity, which can be blocked by the transglycosylase inhibitor moenomycin. Covalently linked to activated Sepharose, PBP1C specifically retained PBP1B and the transpeptidases PBP2 and -3 in addition to the murein hydrolase MltA. The specific interaction with these proteins suggests that PBP1C is assembled into a multienzyme complex consisting of both murein polymerases and hydrolases. Overexpression of PBP1C does not support growth of a PBP1A(ts)/PBP1B double mutant at the restrictive temperature, and PBP1C does not bind to the same variety of penicillin derivatives as PBPs 1A and 1B. Deletion of PBP1C resulted in an altered mode of murein synthesis. It is suggested that PBP1C functions in vivo as a transglycosylase only.     

Characterization of the proteins encoded by the Bacillus subtilis yoxA-dacC operon

In Bacillus subtilis , the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An aldose-1-epimerase or mutarotase activity was measured with the YoxA protein that we propose to rename as GalM by analogy with its counterpart in E. coli . The peptide d -Glu-δ- m -A₂pm- d -Ala- m -A₂pm- d -Ala mimicking the B. subtilis and E. coli interpeptide bridge was synthesized and incubated with the purified dacC product, the PBP4a. A clear dd -endopeptidase activity was obtained with this penicillin-binding protein, or PBP. The possible role of this class of PBP, present in almost all bacteria, is discussed.     

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.     

AmpH, a bifunctional DD-endopeptidase and DD-carboxypeptidase of Escherichia coli

In Escherichia coli, low-molecular-mass penicillin-binding proteins (LMM PBPs) are important for correct cell morphogenesis. These enzymes display DD-carboxypeptidase and/or dd-endopeptidase activities associated with maturation and remodeling of peptidoglycan (PG). AmpH has been classified as an AmpH-type class C LMM PBP, a group closely related to AmpC β-lactamases. AmpH has been associated with PG recycling, although its enzymatic activity remained uncharacterized until now. Construction and purification of His-tagged AmpH from E. coli permitted a detailed study of its enzymatic properties. The N-terminal export signal of AmpH is processed, but the protein remains membrane associated. The PBP nature of AmpH was demonstrated by its ability to bind the β-lactams Bocillin FL (a fluorescent penicillin) and cefmetazole. In vitro assays with AmpH and specific muropeptides demonstrated that AmpH is a bifunctional DD-endopeptidase and DD-carboxypeptidase. Indeed, the enzyme cleaved the cross-linked dimers tetrapentapeptide (D45) and tetratetrapeptide (D44) with efficiencies (k(cat)/K(m)) of 1,200 M(-1) s(-1) and 670 M(-1) s(-1), respectively, and removed the terminal D-alanine from muropeptides with a C-terminal D-Ala-D-Ala dipeptide. Both DD-peptidase activities were inhibited by 40 μM cefmetazole. AmpH also displayed a weak β-lactamase activity for nitrocefin of 1.4 × 10(-3) nmol/μg protein/min, 1/1,000 the rate obtained for AmpC under the same conditions. AmpH was also active on purified sacculi, exhibiting the bifunctional character that was seen with pure muropeptides. The wide substrate spectrum of the DD-peptidase activities associated with AmpH supports a role for this protein in PG remodeling or recycling.     

Overproduction of penicillin-binding protein 2 and its inactive variants causes morphological changes and lysis in Escherichia coli

Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.     

Identification of a penicillin-binding protein 3 homolog, PBP3x, in Pseudomonas aeruginosa: gene cloning and growth phase-dependent expression.

A homolog of Pseudomonas aeruginosa penicillin-binding protein 3 (PBP3), named PBP3x in this study, was identified by using degenerate primers based on conserved amino acid motifs in the high-molecular-weight PBPs. Analysis of the translated sequence of the pbpC gene encoding this PBP3x revealed that 41 and 48% of its amino acids were identical to those of Escherichia coli and P. aeruginosa PBP3s, respectively. The downstream sequence of pbpC encoded convergently transcribed homologs of the E. coli soxR gene and the Mycobacterium bovis adh gene. The pbpC gene product was expressed from the T7 promoter in E. coli and was exported to the cytoplasmic membrane of E. coli cells and could bind [3H] penicillin. By using a broad-host-range vector, pUCP27, the pbpC gene was expressed in P. aeruginosa PAO4089. [3H]penicillin-binding competition assays indicated that the pbpC gene product had lower affinities for several PBP3-targeted beta-lactam antibiotics than P. aeruginosa PBP3 did, and overexpression of the pbpC gene product had no effect on the susceptibility to the PBP3-targeted antibiotics tested. By gene replacement, a PBP3x-defective interposon mutant (strain HC132) was obtained and confirmed by Southern blot analysis. Inactivation of PBP3x caused no changes in the cell morphology or growth rate of exponentially growing cells, suggesting that pbpC was not required for cell viability under normal laboratory growth conditions. However, the upstream sequence of pbpC contained a potential sigma(s) recognition site, and pbpC gene expression appeared to be growth rate regulated. [3H]penicillin-binding assays indicated that PBP3 was mainly produced during exponential growth whereas PBP3x was produced in the stationary phase of growth.     

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.     

Penicillin-binding protein 2x of Streptococcus pneumoniae. Expression in Escherichia coli and purification of a soluble enzymatically active derivative.

A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high-level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl beta-d-thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin-binding capacity. Deletion of 30 hydrophobic N-terminal amino acid residues at positions 19-48 resulted in high-level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full beta-lactam-binding activity. A two-step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.     

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.     

pH,inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5

The recent structural determination of Escherichia coli penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure-function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five alpha- and epsilon-substituted L-Lys-D-Ala-D-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6-12.3, and the k(cat)/K(m) vs. pH profile for activity against Ac(2)-L-Lys-D-Ala-D-Ala was bell-shaped, with pK(a)s at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed DD-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A beta-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.