D-alanine Carboxypeptidase Research Articles

Association of a d -Alanyl- d -Alanine Carboxypeptidase Gene with the Formation of Aberrantly Shaped Cells during the Induction of Viable but Nonculturable Vibrio parahaemolyticus

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that causes human gastroenteritis. When the viable but nonculturable (VBNC) state of this bacterium was induced by incubation at 4°C in Morita minimal salt solution containing 0.5% NaCl, the rod-shaped cells became coccoid, and various aberrantly shaped intermediates were formed in the initial stage. This study examined the factors that influence the formation of these aberrantly shaped cells. The proportion of aberrantly shaped cells was not affected in a medium containing D-cycloserine (50 μg/ml) but was lower in a medium containing cephalosporin C (10 μg/ml) than in the control medium without antibiotics. The proportion of aberrantly shaped cells was higher in a culture medium that contained 0.5% NaCl than in culture media containing 1.0 or 1.5% NaCl. The expression of 15 of 17 selected genes associated with cell wall synthesis was enhanced, and the expression of VP2468 (dacB), which encodes D-alanyl-D-alanine carboxypeptidase, was enhanced the most. The proportion of aberrantly shaped cells was significantly lower in the dacB mutant strain than in the parent strain, but the proportion was restored in the presence of the complementary dacB gene. This study suggests that disturbance of the dynamics of cell wall synthesis by enhanced expression of the VP2468 gene is associated with the formation of aberrantly shaped cells in the initial stage of induction of VBNC V. parahaemolyticus cells under specific conditions.

Psychrobacter arcticus 273-4 Uses Resource Efficiency and Molecular Motion Adaptations for Subzero Temperature Growth

Permafrost soils are extreme environments that exert low-temperature, desiccation, and starvation stress on bacteria over thousands to millions of years. To understand how Psychrobacter arcticus 273-4 survived for >20,000 years in permafrost, transcriptome analysis was performed during growth at 22 degrees C, 17 degrees C, 0 degrees C, and -6 degrees C using a mixed-effects analysis of variance model. Genes for transcription, translation, energy production, and most biosynthetic pathways were downregulated at low temperatures. Evidence of isozyme exchange was detected over temperature for D-alanyl-D-alanine carboxypeptidases (dac1 and dac2), DEAD-box RNA helicases (csdA and Psyc_0943), and energy-efficient substrate incorporation pathways for ammonium and acetate. Specific functions were compensated by upregulation of genes at low temperature, including genes for the biosynthesis of proline, tryptophan, and methionine. RNases and peptidases were generally upregulated at low temperatures. Changes in energy metabolism, amino acid metabolism, and RNase gene expression were consistent with induction of a resource efficiency response. In contrast to results observed for other psychrophiles and mesophiles, only clpB and hsp33 were upregulated at low temperature, and there was no upregulation of other chaperones and peptidyl-prolyl isomerases. relA, csdA, and dac2 knockout mutants grew more slowly at low temperature, but a dac1 mutant grew more slowly at 17 degrees C. The combined data suggest that the basal biological machinery, including translation, transcription, and energy metabolism, is well adapted to function across the growth range of P. arcticus from -6 degrees C to 22 degrees C, and temperature compensation by gene expression was employed to address specific challenges to low-temperature growth.

Crystal Structure of Escherichia coli Penicillin-Binding Protein 5 Bound to a Tripeptide Boronic Acid Inhibitor: A Role for Ser-110 in Deacylation

Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.

Crystal Structure of Wild-type Penicillin-binding Protein 5 from Escherichia coli: IMPLICATIONS FOR DEACYLATION OF THE ACYL-ENZYME COMPLEX

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a d-alanine carboxypeptidase (CPase), cleaving d-alanine from the C terminus of 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 acylenzyme complex (t(1/2) approximately 10 min). A Gly105 --> Asp mutation in PBP 5 markedly impairs deacylation with only minor effects on acylation, and abolishes CPase activity. We have determined the three-dimensional structure of a soluble form of wild-type PBP 5 at 1.85-A resolution and have also refined the structure of the G105D mutant form of PBP 5 to 1.9-A resolution. Comparison of the two structures reveals that the major effect of the mutation is to disorder a loop comprising residues 74-90 that sits atop the SXN motif of the active site. Deletion of the 74-90 loop in wild-type PBP 5 markedly diminished the deacylation rate of penicillin G with a minimal impact on acylation, and abolished CPase activity. These effects were very similar to those observed in the G105D mutant, reinforcing the idea that this mutation causes disordering of the 74-90 loop. Mutation of two consecutive serines within this loop, which hydrogen bond to Ser110 and Asn112 in the SXN motif, had marked effects on CPase activity, but not beta-lactam antibiotic binding or hydrolysis. These data suggest a direct role for the SXN motif in deacylation of the acyl-enzyme complex and imply that the functioning of this motif is modulated by the 74-90 loop.

Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation.

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T(m)) of 48.5 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k(2)/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.

Crystal Structure of a Deacylation-defective Mutant of Penicillin-binding Protein 5 at 2.3-Å 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.

The dacA gene of Bacillus stearothermophilus coding for D-alanine carboxypeptidase: cloning, structure and expression in Escherichia coli and Pichia pastoris.

The bacterial D-alanine carboxypeptidases (CPases) remove C-terminal D-alanyl residues from sugar-peptide cell wall precursors. The CPases have many characteristics in common with the high-M(r) penicillin-binding proteins (PBPs) whose inhibition by beta-lactam antibiotics is lethal. The CPases are attractive as model PBPs, because of their relatively lower M(r) and higher activity in vitro. We have cloned and sequenced the Bacillus stearothermophilus gene (dacA) coding for a membrane-bound CPase. The nucleotide (nt) sequence of the gene is homologous to that of the Escherichia coli and Bacillus subtilis dacA loci, which also code for membrane-bound CPases. E. coli host cells lysed when expression of B. stearothermophilus dacA was induced. The same coding sequence was expressed in the methylotrophic yeast, Pichia pastoris, using the alcohol oxidase-1 (AOX1) promoter. Over 100 micrograms/ml of CPase was efficiently secreted into the medium after induction by methanol, without adversely affecting this host. The yeast product is indistinguishable from the native enzyme in structure and activity. The ability to secrete large amounts of heterologous protein and the lack of endogenous peptidoglycan metabolism makes P. pastoris an attractive candidate for the production of PBPs.

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.

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

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

The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide

The rodA(Sui) mutation allows cell division to take place at 42 degrees C in ftsI23 mutant cells, which produce a thermolabile penicillin-binding protein 3 (PBP3, the septation-specific peptidoglycan transpeptidase). We show here that the mutation in rodA is a single-base change from a glutamine to a chain termination (amber) codon, and that an amber suppressor (supE) present in the strain restores the ability to produce a reduced level of normal RodA protein. The reduced level of RodA is accompanied by an increase in the levels of two other proteins (PBP2 and PBP5) encoded by genes in the rodA operon. We show that an increased level of PBP5 is by itself sufficient to restore cell division to ftsI23 cells at 42 degrees C. Two other treatments were found to restore division capacity to the mutant: an increase in PBP6 (which is a D-alanine carboxypeptidase like PBP5) or suitable concentrations of D-cycloserine. All of the above treatments have the effect of reducing the number of pentapeptide side chains in peptidoglycan and increasing the number of tripeptides. We conclude that the effect of the rodA(Sui) mutation is to indirectly increase the availability of tripeptide side chains, which are used preferentially by PBP3 as acceptors in transpeptidation. A change in the proportions of different kinds of peptide side chain in the peptidoglycan can therefore determine whether cells will divide.

Relations between beta-lactamases and penicillin-binding proteins: beta-lactamase activity of penicillin-binding protein 5 from Escherichia coli.

The molecular mechanism of the hydrolysis of penicillin by penicillin-binding protein (PBP) 5 was investigated. Cysteine-115 had been previously implicated in the hydrolysis of the acyl-enzyme intermediate in PBP5. In studies with oligonucleotide-directed mutagenesis, the role of cysteine-115 in the mechanism of PBP5 was investigated by the mutation of this residue to either a serine or an alanine residue. Genetic truncation of the studied proteins at their carboxyl termini resulted in the production of a soluble form of these proteins (sPBPs). The purified sPBPs were assayed for both their beta-lactamase and their D-alanine carboxypeptidase activities. The results of these assays indicate that the cysteine residue does not directly participate in the enzymatic mechanism. In a further investigation of the catalytic mechanism of beta-lactam hydrolysis, a soluble, naturally occurring mutant was purified in large amounts and crystallized from concentrated polyethylene glycol solutions. This mutant of PBP5 (sPBP5') has a near-normal acylation rate with respect to [14C]penicillin, but shows a drastic increase in the half-life of the acyl-enzyme complex. The crystals of sPBP5' diffract to greater than 3 A resolution and are suitable for detailed crystallographic analysis.

Site-directed mutants of a soluble form of penicillin-binding protein 5 from Escherichia coli and their catalytic properties.

Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated. Alkylation of cysteine 115 with sulfhydryl reagents has previously been shown to inhibit severely the D-alanine carboxypeptidase activity of PBP 5. Alkylation also inhibits the hydrolysis of bound penicillin G, with only a slight effect on its binding. Cysteine 115 in sPBP 5 was changed to either a serine (sPBP 5C-S) or an alanine (sPBP 5C-A) residue. The wild-type and mutant sPBPs were purified in milligram amounts from induced cultures by ampicillin affinity chromatography. The mutant PBPs showed only a 2-fold increase in the half-life of the penicilloyl-PBP complex, and had a binding affinity for penicillin G identical to wild-type PBP 5. The Km for the release of D-alanine from the peptide L-Ala-D-gamma-Glu-L-Lys-D-Ala-D-Ala was 5.0, 3.5, and 7.8 mM for PBP 5, PBP 5C-S, and PBP 5C-A, respectively, while the values for Vmax were 2.5, 3.3, and 5.1 mumol/min/mg. From these data it was concluded that the cysteine residue does not directly participate in the enzymatic mechanism.

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.

Mutation in Pseudomonas aeruginosa causing simultaneous defects in penicillin-binding protein 5 and in enzyme activities of penicillin release and D-alanine carboxypeptidase.

Penicillin-binding protein 5 in Pseudomonas aeruginosa had moderately penicillin-sensitive D-alanine carboxypeptidase activity. As in Escherichia coli, a defect in this enzyme activity was not lethal.

Sequences of the active-site peptides of three of the high-Mr penicillin-binding proteins of Escherichia coli K-12.

The amino acid compositions of the radioactive peptides obtained from trypsin digestion of [14C]benzylpenicillin-labeled penicillin-binding proteins (PBPs) 1A, 1B, and 3 of Escherichia coli have been obtained. Complete digestion of these peptides with a combination of aminopeptidase M and carboxypeptidase Y showed that benzylpenicillin was bound to a serine residue in each of these proteins. Comparison of the compositions of the penicillin-labeled peptides with the complete amino acid sequences of PBPs 1A, 1B, and 3 showed that the acylated serine occurs near the middle of each of the proteins, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The sequence around the acylated serine of these high Mr PBPs shows little similarity to that around the acylated serine of the low-Mr PBPs (D-alanine carboxypeptidases) or of the class A or class C beta-lactamases, except that in all of these enzymes which interact with penicillin the acylated serine residue occurs within the sequence Ser-Xaa-Xaa-Lys.

A direct spectrophotometric assay for d-alanine carboxypeptidases and for the esterase activity of β-lactamases

A direct spectrophotometric pH indicator method has been devised to assay the activity of the d-Ala carboxypeptidase/transpeptidase of Streptomyces R61, and which should be of general application to d-Ala carboxypeptidases. The substrate employed is N,N′-diacetyl- l-lysyl- d-alanyl- d-lactate. The method allows the determination of steady-state kinetic parameters, and can also be used for the assay of the esterase activity of β-lactamases against specific depsipeptides.

Purification and properties of penicillin-binding proteins 5 and 6 from the dacA mutant strain of Escherichia coli (JE 11191).

Penicillin-binding proteins 5 and 6 have been purified to homogeneity from the dacA mutant strain of Escherichia coli (JE 11191). Protein 6 from the mutant strain appears to be identical to that from the wild type, but protein 5 is a mutant protein which has no D-alanine carboxypeptidase activity. Moreover, the mutant protein 5 binds, but does not release, [14C]penicillin G. Correspondingly, an acyl-enzyme intermediate derived from a synthetic substrate is accumulated by the mutant protein. A comparison of the acylation site for the synthetic substrate and for penicillin G by limited proteolysis and some other properties of the mutant protein are described.

Studies on the mechanism of action of imipenem (N-formimidoylthienamycin) in vitro: binding to the penicillin-binding proteins (PBPs) in Escherichia coli and Pseudomonas aeruginosa, and inhibition of enzyme activities due to the PBPs in E. coli.

The binding affinities of imipenem (N- formimidoylthienamycin ) to penicillin-binding proteins ( PBSs ) of Escherichia coli and Pseudomonas aeruginosa were determined by two different methods in which competition with [14C]benzylpenicillin for the binding sites was measured. By both methods imipenem was shown to have very high binding affinities to PBPs-2 and -4 in E. coli and P. aeruginosa, and appreciable affinities to most of their other major PBPs. But higher concentrations of imipenem were required for binding to the PBPs-3 in these bacteria. More direct information about the antibacterial activity of imipenem was obtained by measuring its inhibition of the peptidoglycan-synthetic enzyme activities of E. coli PBPs. The results of enzyme inhibitions were compatible with those obtained in binding experiments. The antibiotic inhibited the transpeptidase activities of PBPs-1A, -1B and -2, and the D-alanine carboxypeptidase activities of PBPs-4 and -5. The antibiotic also seemed to cause strong inhibition of the transglycosylase activity of PBP-1A by some unknown mechanism. It inhibited the transpeptidase activity of PBP-3 only weakly, which is consistent with the findings that it had low binding affinity to PBP-3 and did not inhibit septum formation by the cells.

Murein structure and lack of DD- and LD-carboxypeptidase activities in Caulobacter crescentus.

High-pressure liquid chromatography of a muramidase digest of murein sacculi from Caulobacter crescentus showed that the absence of D-alanine carboxypeptidase activity in the cells was reflected by a very high content of pentapeptide in the murein. Approximately half of the pentapeptide side chains were shown to contain glycine, which replaced D-alanine as the terminal amino acid.

Synthesis of penicillin-binding protein 6 by stationary-phase Escherichia coli.

The level of penicillin-binding protein 6, a D-alanine carboxypeptidase I, was found to be 2- to 10-fold higher in stationary-phase cells than in exponentially growing cells of Escherichia coli. This increase appeared to be due to de novo synthesis rather than to an unmasking of preexisting material. There was no comparable change in the amount of any of the other six penicillin-binding proteins.