Catalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process.     

Primary structure of the Streptomyces R61 extracellular DD-peptidase. 1. Cloning into Streptomyces lividans and nucleotide sequence of the gene

An 11,450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptomyces R61 was cloned in Streptomyces lividans using the high-copy-number plasmid pIJ702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DD-peptidase, this precursor possesses a cleavable 31-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD-peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD-peptidase with those of several active-site serine beta-lactamases and penicillin-binding proteins of Escherichia coli shows homology in those sequences that comprise the active-site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD-peptidase/Escherichia coli ampC beta-lactamase (class C). Since the Streptomyces R61 DD-peptidase and beta-lactamases of class A have very similar three-dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429-1431; Samraoui et al. (1986) Nature (Lond.) 320, 378-380], it is concluded that these tertiary features are probably also shared by the beta-lactamases of class C, i.e. that the Streptomyces R61 DD-peptidase and the beta-lactamases of classes A and C are related in an evolutionary sense.     

Understanding the acylation mechanisms of active-site serine penicillin-recognizing proteins: a molecular dynamics simulation study

Beta-lactam antibiotics inhibit enzymes involved in the last step of peptidoglycan synthesis. These enzymes, also identified as penicillin-binding proteins (PBPs), form a long-lived acyl-enzyme complex with beta-lactams. Antibiotic resistance is mainly due to the production of beta-lactamases, which are enzymes that hydrolyze the antibiotics and so prevent them reaching and inactivating their targets, and to mutations of the PBPs that decrease their affinity for the antibiotics. In this study, we present a theoretical study of several penicillin-recognizing proteins complexed with various beta-lactam antibiotics. Hybrid quantum mechanical/molecular mechanical potentials in conjunction with molecular dynamics simulations have been performed to understand the role of several residues, and pK(a) calculations have also been done to determine their protonation state. We analyze the differences between the beta-lactamase TEM-1, the membrane-bound PBP2x of Streptococcus pneumoniae, and the soluble DD-transpeptidase of Streptomyces K15.     

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.     

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.     

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.     

Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.     

The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family.

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments.     

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.     

Penicillin-binding protein 4 of Escherichia coli shows a novel type of primary structure among penicillin-interacting proteins

The nucleotide sequence of a 1884 bp DNA fragment of E. coli, carrying the gene dacB, was determined. The DNA codes for penicillin-binding protein 4 (PBP4), an enzyme of 477 amino acids, being involved as a DD-carboxypeptidase-endopeptidase in murein metabolism. The enzyme is translated with a cleavable signal peptide of 20 amino acids, which was verified by sequencing the amino-terminus of the isolated protein. The characteristic active-site fingerprints SXXK, SXN and KTG of class A beta-lactamases and penicillin-binding proteins were located in the sequence. On the basis of amino acid alignments we propose, that PBP4 and class A beta-lactamases share a common evolutionary origin but PBP4 has acquired an additional domain of 188 amino acids in the region between the SXXK and SXN elements.     

The ddcA gene from Streptomyces fradiae encodes an extracellular beta-lactamase with penicillinase and cephalosporinase activities

The ddcA gene from Streptomyces fradiae, which is located adjacent to the left edge of the tylosin biosynthetic cluster, has been cloned and sequenced. DNA sequence analysis revealed an ORF of 1194 bp that encodes a product of 42.6 kDa. This protein showed significant similarity to the extracellular endopeptidase with beta-lactamase activity encoded by the adp gene from Bacillus cereus and to PBPs (DD-carboxypeptidases and DD-endopeptidases) and beta-lactamases. Moreover, it contains three characteristic motifs conserved in PBPs and beta-lactamases, including an essential serine residue in the active centre and a putative leader peptide. Heterologous expression of the ddcA gene in Streptomyces lividans demonstrated the presence in the transformants of an extracellular beta-lactamase active against penicillin G, ampicillin and the chromogenic cephalosporin nitrocefin.     

Crystal structure of a deacylation-defective mutant of penicillin-binding protein 5 at 2.3-A resolution

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a d-alanine carboxypeptidase, cleaving the C-terminal d-alanine residue from cell wall peptides. Like all PBPs, PBP 5 forms a covalent acyl-enzyme complex with beta-lactam antibiotics; however, PBP 5 is distinguished by its high rate of deacylation of the acyl-enzyme complex (t(12) approximately 9 min). A Gly-105 --> Asp mutation in PBP 5 markedly impairs this beta-lactamase activity (deacylation), with only minor effects on acylation, and promotes accumulation of a covalent complex with peptide substrates. To gain further insight into the catalytic mechanism of PBP 5, we determined the three-dimensional structure of the G105D mutant form of soluble PBP 5 (termed sPBP 5') at 2.3 A resolution. The structure is composed of two domains, a penicillin binding domain with a striking similarity to Class A beta-lactamases (TEM-1-like) and a domain of unknown function. In addition, the penicillin-binding domain contains an active site loop spatially equivalent to the Omega loop of beta-lactamases. In beta-lactamases, the Omega loop contains two amino acids involved in catalyzing deacylation. This similarity may explain the high beta-lactamase activity of wild-type PBP 5. Because of the low rate of deacylation of the G105D mutant, visualization of peptide substrates bound to the active site may be possible.     

Beta-lactamase of Bacillus licheniformis 749/C. Refinement at 2 A resolution and analysis of hydration

The crystallographic and molecular structure of the class A beta-lactamase (penicillinase) of Bacillus licheniformis 749/C has been refined with X-ray diffraction data to 2 A resolution. For the 27,330 data with F greater than or equal to 3 sigma(F), the R factor is 0.15; for all 30,090 data, R is 0.16. The estimated co-ordinate error is 0.15 A. In the final model, the deviation of covalent bonds and angles from ideality is 0.012 A and 2.2 degrees, respectively. The model includes two molecules of 29,500 daltons each in the asymmetric unit of space group P2(1), 484 water molecules and two tetrahedral buffer anions. Overlay of the two protein molecules results in a root-mean-square difference of 0.17 A and 0.41 A for alpha-carbon atoms and for all atoms, respectively. Twenty-six water molecules fall within 0.25 A of matching water molecules associated with the second protein molecule. The reactive Ser70 is on a turn of 3(10) helix at the N terminus of a longer alpha-helix (72-83). The penicillin-binding site near this helix contains at least seven water molecules. Upon penicillin entry, a water molecule in the oxyanion hole, hydrogen-bonded between the N terminus of helix (80-83) and beta-strand (230-238), would be displaced by the oxygen atom of the beta-lactam carbonyl group. An unexpelled molecule of water is proposed to be the catalytic water required for penicillin hydrolysis. The water is hydrogen-bonded to Glu166, a conserved residue in all beta-lactamases, and it lies 3 A from the alpha-face of a previously modeled penicillin. The position of the water-Glu166 pair is stabilized in the active site by a cis peptide bond at Pro167.     

Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl.

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.     

Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent.

The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-alpha and alpha/beta folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK D,D-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4-->3) type. They are inactivated by penicillin and other beta-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4-->3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze D,D peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine beta-lactamases, and the penicillin sensors of several penicillin sensory transducers help the D,D-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Pen(r) protein fusions. Plausible hypotheses are put forward on the roles that the Pen(r) protein fusions, acting as L,D-acyltransferases, may play in the (3-->3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4-->3) type to the (3-->3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant.     

Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding.

All penicillin-binding proteins (PBPs) contain a conserved box of homology in the carboxyl-terminal half of their primary sequence that can be Lys-Thr-Gly, Lys-Ser-Gly, or His-Thr-Gly. Site-saturation mutagenesis was used to address the role of the lysine residue at this position (Lys213) in Escherichia coli PBP 5, a D-alanine carboxypeptidase enzyme. A soluble form of PBP 5 was used to replace Lys213 with 18 other amino acids, and the ability of these mutant proteins to bind [3H]penicillin G was assessed. Only the substitution of lysine with arginine resulted in a protein that was capable of forming a stable covalent complex with antibiotic. The affinity of [14C]penicillin G for the arginine mutant was 1.2-fold higher than for wild-type PBP 5 (4.4 versus 5.1 micrograms/ml for 20 min at 30 degrees C), and both proteins showed identical rates of hydrolysis of the [14C]penicilloyl-bound complex (t1/2 = 9.1 min). Surprisingly, the arginine-substituted protein was unable to catalyze D-alanine carboxypeptidase activity in vitro, which suggests that there is a substantial difference in the geometries of the peptide substrate and penicillin G within the active site of PBP 5.     

The importance of a critical protonation state and the fate of the catalytic steps in class A beta-lactamases and penicillin-binding proteins

Beta-lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222)

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.     

Crystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the 'penicillin-recognizing enzyme' family

BACKGROUND: beta-Lactam compounds are the most widely used antibiotics. They inactivate bacterial DD-transpeptidases, also called penicillin-binding proteins (PBPs), involved in cell-wall biosynthesis. The most common bacterial resistance mechanism against beta-lactam compounds is the synthesis of beta-lactamases that hydrolyse beta-lactam rings. These enzymes are believed to have evolved from cell-wall DD-peptidases. Understanding the biochemical and mechanistic features of the beta-lactam targets is crucial because of the increasing number of resistant bacteria. DAP is a D-aminopeptidase produced by Ochrobactrum anthropi. It is inhibited by various beta-lactam compounds and shares approximately 25% sequence identity with the R61 DD-carboxypeptidase and the class C beta-lactamases. RESULTS: The crystal structure of DAP has been determined to 1.9 A resolution using the multiple isomorphous replacement (MIR) method. The enzyme folds into three domains, A, B and C. Domain A, which contains conserved catalytic residues, has the classical fold of serine beta-lactamases, whereas domains B and C are both antiparallel eight-stranded beta barrels. A loop of domain C protrudes into the substrate-binding site of the enzyme. CONCLUSIONS: Comparison of the biochemical properties and the structure of DAP with PBPs and serine beta-lactamases shows that although the catalytic site of the enzyme is very similar to that of beta-lactamases, its substrate and inhibitor specificity rests on residues of domain C. DAP is a new member of the family of penicillin-recognizing proteins (PRPs) and, at the present time, its enzymatic specificity is clearly unique.     

Production of thiol-penicillin-binding protein 3 of Escherichia coli using a two primer method of site-directed mutagenesis.

The active site serine residue of penicillin-binding protein 3 of Escherichia coli that is acylated by penicillin (Ser-307) has been converted to a cysteine residue using a simple and efficient two primer method of site-directed mutagenesis. The resulting thiol-penicillin-binding protein 3 was expressed under the control of the lacUV5 promoter in a high copy number plasmid. Constitutive expression of the thiol-enzyme (but not of the wild-type enzyme) was lethal, and the plasmid could only be maintained in E. coli strains that carried the lacIq mutation. Induction of the expression of the thiol-enzyme resulted in inhibition of cell division and the growth of the bacteria into very long filamentous cells. The inhibition of septation was probably due to interference of the function of the wild-type penicillin-binding protein 3 in cell division by the enzymatically inactive thiol-enzyme, and this implies that penicillin-binding protein 3 acts as part of a complex in vivo. We were unable to detect any acylation of the thiol-enzyme by penicillin, but it is not yet clear if this was because the thioester was not formed at an appreciable rate, or if it was formed but was too unstable to be detected by a modified penicillin-binding protein assay.