Biomedical subjects

W Keck

Publications and source records attributed to W Keck.

At least 55 records · Page 3Linked to original sources

PubMed1992

Cytoplasmic high-level expression of a soluble, enzymatically active form of the Escherichia coli penicillin-binding protein 5 and purification by dye chromatography.

High-level expression of a soluble form of penicillin-binding protein 5 (PBP5), called PBP5s, and translocation across the cytoplasmic membrane results in lysis of Escherichia coli cells. The detrimental effect of increased amounts of this D,D-carboxypeptidase on the stability of murein polymer can be avoided by accumulation of the overexpressed protein in the cytoplasm. The signal peptide of the structural gene dacAs, coding for PBP5s was deleted by creating a BamHI site at the site of processing and the truncated gene dacAsc was cloned under the control of the lambda PR promoter. Temperature induction resulted in a 200-fold overproduction of the mature PBP5s in the cytosol (PBP5sc) which is no longer harmful to the cells. PBP5sc could quantitatively be recovered in the soluble fraction after disrupting the cells. The protein retained full enzymatic activity as measured by the release of D-alanine from bisacetyl-L-Lys-D-Ala-D-Ala and formation of [14C]penicillin-protein complex at a 1:1 stoichiometry. A one-step purification procedure using the immobilized dye Procion rubine MX-B resulted in homogeneous preparations of both wild-type and mutated forms of PBP5sc.

Alanine↗

PubMed1992

Enzymatic preparation of 1,6-anhydro-muropeptides by immobilized murein hydrolases from Escherichia coli fused to staphylococcal protein A.

In order to produce biologically active 1,6-anhydro-muropeptides in large amounts by enzymatic degradation of isolated bacterial murein polymer highly specific periplasmic murein-metabolizing enzymes from Escherichia coli are made available. The genes slt, dacB, and mepA, encoding the soluble lytic transglycosylase (Slt), the penicillin-sensitive DD-endopeptidase (PBP4), and the penicillin-insensitive murein endopeptidase A (MepA), were independently fused to the N-terminal encoding sequence of staphylococcal protein A (SpA) under control of the temperature-inducible phage lambda pR promoter. The SpA fusion proteins were stably over-produced at high levels in E. coli upon temperature induction at 42 degrees C and account for 3% (5 mg SpASlt/l culture), 3% (5 mg SpAPBP4/l culture), and 0.3% (0.5 mg SpAMepA/l culture) of total protein. The SpA fusion proteins, immobilized on IgG Sepharose, are proteolytically sensitive, in vitro, resulting in complete degradation of the SpA portion of the fusion proteins and release of the murein hydrolases in intact and enzymatically active form into the supernatant. Proteolytic degradation could be prevented by p-hydroxymercuribenzoic acid (PHMB) or ethylenediaminetetraacetate (EDTA) suggesting the involvement of the periplasmic protease Pi from E. coli. The immobilized fusion proteins were enzymatically active and could be used for the batch production of biologically active 1,6-anhydro-muropeptides, which were successively separated on HPLC. Isolated murein polymer was degraded quantitatively to monomeric 1,6-anhydro-muropeptides when immunoglobulin G (IgG)-SpASlt was used in combination with IgG-SpAMepA. A combination of IgG-SpASlt with IgG-SpAPBP4 left the 1,6-anhydro-dimers and oligomers being cross-linked via an LD-peptide bond (m-DAP-m-DAP) uncleaved.

Amino Acid Sequence↗

PubMed1992

Rapid screening of a large number of immobilized textile dyes for the purification of proteins: use of penicillin-binding protein 4 of Escherichia coli as a model enzyme.

A rapid method for screening the affinity of proteins to dye-modified resins is described. Performing the binding and elution of the protein extracts in a batch-wise manner and eluting the bound proteins with SDS-PAGE denaturation buffer speed up the screening process and allow the analysis of large collections of dyes. Penicillin-binding protein 4 of Escherichia coli was used as a model enzyme to determine the influences of pH, metal ions, and ionic strength (0 to 500 mM NaCl) on its binding behavior using a collection of 98 dye-affinity resins.

Bacterial Proteins↗

PubMed1992

Deletion of an additional domain located between SXXK and SXN active-site fingerprints in penicillin-binding protein 4 from Escherichia coli.

It was suggested previously that the primary structure of penicillin-binding protein 4 (PBP4) is new and unique among proteins that interact with penicillin. Our proposal that PBP4 carries an additional domain, located between the active-site fingerprints SXXK and SXN, was investigated by mutational deletion analysis. A clustered set of internal deletions was created in this region by exonuclease treatment of the dacB coding DNA, starting from two internal restriction sites. PBP4 mutants carrying internal deletions were selected by screening for immunoreactive forms of PBP4 with reduced molecular weight that were still active with respect to penicillin binding. DNA sequencing revealed 24 distinct PBP4 mutants with internal deletions ranging from 37 to 113 amino acids. The amino- and carboxy-terminal end points of the deletions were not randomly distributed but tended to cluster in certain areas. Overproduction of the individual mutated forms of PBP4 resulted in accumulation of the major portion of the proteins in the particulate cell fraction. The yield of soluble and active mutated forms of the protein was reduced from below 1% to 79% of the level obtained for the native protein. The deletions that were introduced had minor effects on the deacylation rate of bound benzylpenicillin. Two pairs of cysteine residues (Cys-139-Cys-153 and Cys-197-Cys-214) that are located in the deletable region may form disulfide bridges.

Bacterial Proteins↗

PubMed1992

Murein-metabolizing enzymes from Escherichia coli: existence of a second lytic transglycosylase.

In addition to the soluble lytic transglycosylase, a murein-metabolizing enzyme with a molecular mass of 70 kDa (Slt70), Escherichia coli possesses a second lytic transglycosylase, which has been described as a membrane-bound lytic transglycosylase (Mlt; 35 kDa; EC 3.2.1.-). The mlt gene, which supposedly encodes Mlt, was cloned, and the complete nucleotide sequence was determined. The open reading frame, identified on a 1.7-kb SalI-PstI fragment, codes for a protein of 323 amino acids (M(r) = 37,410). Two transmembrane helices and one membrane-associated helix were predicted in the N-terminal half of the protein. Lysine and arginine residues represent up to 15% of the amino acids, resulting in a calculated isoelectric point of 10.0. The deduced primary structure did not show significant sequence similarity to Slt70 from E. coli. High-level expression of the presumed mlt gene was not paralleled by an increase in murein hydrolase activity. To clarify the identity of the second transglycosylase, we purified an enzyme with the specificity of a transglycosylase from an E. coli slt deletion strain. The completely soluble transglycosylase, with a molecular mass of approximately 35 kDa, was designated Slt35. Its determined 26 N-terminal amino acids showed similarity to a segment in the middle of the Slt70 primary structure. Polyclonal anti-Mlt antibodies, which had been used for the isolation of the mlt gene, were found to cross-react with Mlt as well as with Slt35, suggesting that the previously described Mlt preparation was contaminated with Slt35. We conclude that the second transglycosylase of E. coli is not a membrane-bound protein but rather is a soluble protein.

Amino Acid Sequence↗

PubMed1992

Possible role of Escherichia coli penicillin-binding protein 6 in stabilization of stationary-phase peptidoglycan.

Plasmids for high-level expression of penicillin-binding protein 6 (PBP6) were constructed, giving rise to overproduction of PBP6 under the control of the lambda pR promoter in either the periplasmic or the cytoplasmic space. In contrast to penicillin-binding protein 5 (PBP5), the presence of high amounts of PBP6 in the periplasm as well as in the cytoplasm did not result in growth as spherical cells or in lysis. Deletion of the C-terminal membrane anchor of PBP6 resulted in a soluble form of the protein (PBP6s350). Electron micrographs of thin sections of cells overexpressing both native membrane-bound and soluble PBP6 in the periplasm revealed a polar retraction of the cytoplasmic membrane. Cytoplasmic overexpression of native PBP6 gave rise to the formation of membrane vesicles, whereas the soluble PBP6 formed inclusion bodies in the cytoplasm. Both the membrane-bound and the soluble forms of PBP6 were purified to homogeneity by using the immobilized dye Procion rubine MX-B. Purified preparations of PBP6 and PBP6s350 formed a 14[C]penicillin-protein complex at a 1:1 stoichiometry. The half-lives of the complexes were 8.5 and 6 min, respectively. In contrast to PBP5, no DD-carboxypeptidase activity could be detected for PBP6 by using bisacetyl-L-Lys-D-Ala-D-Ala and several other substrates. These findings led us to conclude that PBP6 has a biological function clearly distinct from that of PBP5 and to suggest a role for PBP6 in the stabilization of the peptidoglycan during stationary phase.

Amino Acid Sequence↗

PubMed1992

A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm of Escherichia coli.

A vector encoding the Staphylococcal protein A was modified by cloning the spa gene, including its signal peptide-encoding sequence, downstream of the translation initiation sites of the phage lambda cro gene and under the control of the temperature-inducible phage lambda pR promoter. The expression from this construct was studied using the Escherichia coli phoA gene as a reporter gene after fusion to the spa gene. Determination of alkaline phosphatase activity, 1 h after temperature induction of expression at 42 degrees C, revealed an 800-fold increase over host strain background level. The presence of the alternating selectable markers on the described vector, pHEMa153, which are essential for efficient oligonucleotide-directed construction of mutations by the gapped duplex DNA method, allows the construction of recombinant and mutated forms of Staphylococcal protein A fusion proteins and efficient expression of spa gene fusions without changing the vector system.

Amino Acid Sequence↗

PubMed1991

Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins.

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.

Acyltransferases↗

PubMed1991

Cytoplasmic phosphorylating domain of the mannitol-specific transport protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli: overexpression, purification, and functional complementation with the mannitol binding domain.

The cytoplasmic C-terminal domain, residues 348-637, and the membrane-bound N-terminal domain, residues 1-347, of EIImtl have been subcloned and expressed in Escherichia coli. The N-terminal domain, IICmtl, contains the mannitol binding site, and the C-terminal domain, IIBAmtl, contains the activity-linked phosphorylation sites, His-554 and Cys-384. Overexpression of the BA domain was achieved by a translational in-frame fusion of the gene with the cro ATG start codon, downstream of the strong PR promoter of phage lambda. The domain has been purified and characterized in in vitro complementation assays. It possessed no mannitol phosphorylation activity itself but was able to restore the phosphoenolpyruvate-dependent phosphorylation activity of two EIImtl phosphorylation site mutants, lacking His-554 or Cys-384. The complementary N-terminal domain was also expressed. Membranes possessing IICmtl were unable to phosphorylate mannitol at the expense of phosphoenolpyruvate. However, when the membranes were combined with the purified C-terminal domain, mannitol phosphorylation activity was restored. Mannitol transport and phosphorylation were also restored in vivo when the two plasmids encoding the N- and C-terminal domains were expressed in the same cell. These data demonstrate the existence of structurally and functionally distinct domains in EIImtl: a cytoplasmic domain with phosphorylating activity and a membrane-bound N-terminal domain which, in the presence of the cytoplasmic domain, is able to actively transport and phosphorylate mannitol. The ability to separate, overproduce, and purify structurally stable, enzymatically active domains opens the way for 3D structural studies as well as complete kinetic analysis of the activities of the individual domains and their interactions.

Base Sequence↗

PubMed1991

Purification of penicillin-binding protein 4 of Escherichia coli as a soluble protein by dye-affinity chromatography.

The dacB gene of Escherichia coli, coding for penicillin-binding protein 4 (PBP4) was cloned under the control of the phage lambda pR promoter and cro gene translation signals. Depression of the phage lambda promoter for 2 h at 42 degrees C in E. coli led to the maximum over-production of PBP4 to 3.8% of the total soluble protein. Expression at 42 degrees C but not at 40 degrees C or 37 degrees C led to incomplete processing and aggregation of the preform of PBP4. Cibacron navyblue 2G-E was selected from a collection of triazine dyes as having a high affinity for PBP4. The immobilised dye was used in a two-step procedure to isolated 374 mg PBP4 from the soluble fraction of 125 g (wet mass) cells of the over-producing strain, with a recovery of 63.2% and a final purity of 99% as determined by active-site titration with radiolabelled penicillin. Saturation of PBP4 with various beta-lactam derivatives did not abolish binding to the dye material, nor was PBP4 eluted by addition of beta-lactams from the dye matrix. PBP4 behaved as a soluble protein throughout the purification, that was performed in the complete absence of detergents. Furthermore, in flotation experiments on sucrose density gradients and in Triton X-114 fractionation experiments, it showed the characteristics of a soluble protein. Cibacron navyblue 2G-E showed class specificity for all E. coli PBP except PBP3 and could be used for the isolation of these PBP from membrane extracts.

Bacterial Proteins↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1991

Details of mannitol transport in Escherichia coli elucidated by site-specific mutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system.

The mannitol transport protein (EIImtl) carries out translocation with concomitant phosphorylation of mannitol from the periplasm to the cytoplasm, at the expense of phosphoenolpyruvate (PEP). The phosphoryl group which is needed for this group translocation is sequentially transferred from PEP via two phosphorylation sites, located exclusively on the C-terminal cytoplasmic domain, to mannitol. Oligonucleotide-directed mutagenesis was used to investigate the precise role of these sites in phosphoryl group transfer, by producing specific amino acid substitutions. The first phosphorylation site, His-554 (P1), was replaced by Ala, which renders the EII-H554A completely inactive in PEP-dependent mannitol phosphorylation, but not in mannitol/mannitol 1-phosphate exchange. The P2 site mutant, EII-C384S, was inactive both in the mannitol phosphorylation reaction and in the exchange reaction, due to replacement of the essential Cys-384 by Ser. Although EII-H554A and EII-C384S were both catalytically inactive in the PEP-dependent phosphorylation, EII-C384S was able to restore up to 55% of the wild-type mannitol phosphorylation activity with the EII-H554A mutant, indicating a direct phosphotransfer between two subunits. These phosphorylation data together with the data obtained from mannitol/mannitol phosphate exchange kinetics, after mixing EII-H554A and EII-C384S, indicated the formation of functionally stable heterodimers, which consist of an EII-H554A and an EII-C384S monomer.

Base Sequence↗

PubMed1991

Phosphoenolpyruvate-dependent mannitol phosphotransferase system of Escherichia coli: overexpression, purification, and characterization of the enzymatically active C-terminal domain of enzyme IImtl equivalent to enzyme IIImtl.

The extreme C-terminus (Ser-490 to Lys-637) of the Escherichia coli EIImtl was subcloned to test structural and mechanistic proposals about the existence of an EIII-like domain in this enzyme. Oligonucleotide-directed mutagenesis was used to produce a unique NcoI restriction site and, at the same time, to change Ser-490 into methionine in a flexible region in front of the proposed EIII-like domain. The 16-kDa C-terminal domain (CI) was overexpressed in Escherichia coli, purified, and analyzed in vitro for catalytic activity in the presence of an EIImtl mutated at its first phosphorylation site, His-554 (EII-H554A). The results presented show that this domain can be expressed as a structurally stable, enzymatically active entity which is able to restore the PEP-dependent phosphorylation activity of the mutant EIImtl-H554A to 25% of wild-type levels. To demonstrate the EIII activity of the CI domain in a more direct way, we also substituted it for EIIImtl in the Staphylococcus carnosus system. The CI domain was active in transferring the phosphoryl group to Staph. carnosus EII; however, it was 6.5 times less active compared to Staph. carnosus EIIImtl itself. EIIImtl from Staph. carnosus, on the other hand, was able to substitute for the isolated C-terminal domain in the E. coli mannitol phosphorylation assay; however, it appeared to be 2 or 3 times less effective.

Base Sequence↗

PubMed1991

Penicillin-binding protein 4 of Escherichia coli: molecular cloning of the dacB gene, controlled overexpression, and alterations in murein composition.

The penicillin-binding protein 4 (PBP4), from Escherichia coli, a DD-carboxypeptidase/DD-endopeptidase, was purified in an enzymatically active form to homogeneity by affinity chromatography on 6-aminopenicillanic acid/Sepharose and heparin/Sepharose. Polyclonal antibodies raised against the pure protein were used to identify and isolate PBP4 overproducing clones from an E. coli expression library, which was established on the basis of a temperature-inducible runaway replication plasmid. Three positive clones were isolated, one of which carried the intact structural gene dacB that codes for PBP4, on a 1.9kb SmaI-EcoRI fragment, whereas the other two carried truncated forms of this gene. The direction of transcription was determined. The PBP4 overproducing strain, when grown in rich medium, tolerated 160-fold overexpression. After disrupting cells by sonication, the majority (80%) of the overproduced PBP4 was detected in the 100,000 X g supernatant. Southern blotting analysis using the cloned dacB gene as a probe revealed that, in contrast to that described by Takeda et al. (1981), the plasmid pLC18-38 of the Clarke-Carbon collection does not code for PBP4. The overall composition of murein, synthesized in vitro or in vivo by the PBP4 overproducing strain, as determined by high-performance liquid chromatography analysis, suggests that PBP4 is not involved in transpeptidation but exclusively catalyses a DD-carboxypeptidase and DD-endopeptidase reaction.

Bacterial Proteins↗

PubMed1991

Murein-metabolizing enzymes from Escherichia coli: sequence analysis and controlled overexpression of the slt gene, which encodes the soluble lytic transglycosylase.

The complete nucleotide sequence of the slt gene encoding the soluble lytic transglycosylase (Slt; EC 3.2.1.-) from Escherichia coli has been determined. The largest open reading frame identified on a 2.5-kb PvuII-SalI fragment indicates that the enzyme is translated as a preprotein of either 654 or 645 amino acids, depending on which of two potential start codons is used. The two possible translation products differ only in the lengths of their predicted signal peptides, 36 or 27 amino acids, respectively. In both cases, processing results in a soluble mature protein of 618 amino acids (Mr = 70,468). The deduced primary structure of the mature protein was confirmed by N-terminal sequencing and determination of the amino acid composition of the isolated transglycosylase. The slt gene contains a high percentage of rare codons, comparable to other low-expressed genes. A hairpin structure that could serve as a transcriptional terminator is located downstream of the slt coding region and precedes the trpR open reading frame at 99.7 min on the E. coli chromosomal map. A computer-assisted search did not reveal any significant sequence similarity to other known carbohydrate-degrading enzymes, including lysozymes. Interestingly, a stretch of 151 amino acids at the C terminus of the transglycosylase shows similarity to the N-terminal portion of the internal virion protein D from bacteriophage T7. Overexpression of the slt gene, under the control of the temperature-inducible phage lambda pR promoter, results in a 250-fold overproduction of the mature transglycosylase, whereas after deletion of the signal peptide a 100-fold overproduction of the enzyme is observed in the cytoplasm.

Amino Acid Sequence↗

PubMed1990

Crystallization of the soluble lytic transglycosylase from Escherichia coli K12.

Lytic transglycosylases degrade the murein polymer of the bacterial cell wall to 1,6-anhydromuropeptides. These enzymes are of significant medical interest, not only because they are ideal targets for the development of new classes of antibiotics, but also because the low molecular weight products of their catalytic action can cause diverse biological activities in humans, which can be either beneficial or toxic. A soluble lytic transglycosylase was purified from an overproducing Escherichia coli strain and X-ray quality crystals were obtained at room temperature from hanging drops by vapor diffusion against 20 to 25% (NH4)2SO4, in 100 mM-sodium acetate buffer, pH 5.0. The crystals diffract in the X-ray beam to 2.8 A resolution. Their space group is P2(1)2(1)2(1) with cell dimensions a = 81 A, b = 88 A and c = 135 A. Assuming one monomer (Mr 70,362) per asymmetric unit, the solvent content of these crystals is 63%.

Escherichia coli↗

PubMed1990

Control of the activity of the soluble lytic transglycosylase by the stringent response in Escherichia coli.

The soluble lytic transglycosylase (Slt) of Escherichia coli is known to be a powerful murein hydrolase in vitro. It is shown here to act as an autolysin in vivo as well. Rapid autolysis of Slt overproducing cells was induced by protein biosynthesis inhibitors, which also block the fomration of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). When amino acid starvation was used to inhibit protein synthesis, autolysis was suppressed in relA+ but not in relA- cells. These findings indicate that the stringent control modulates the enzymatic activity of the soluble lytic transglycosylase in vivo.

Amino Acids↗

PubMed1990

Regulated high-level expression of the mannitol permease of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Escherichia coli.

The structural gene (mtlA) of the Escherichia coli phosphoenolpyruvate-dependent mannitol-transport protein (EIImtl) and its upstream promoter region (Pmtl) were subcloned approximately 150 base pairs downstream of a lambda PR promoter on a multicopy mutagenesis/expression vector and used to transform a mutant (MtlA-) E. coli strain. Induction at 42 degrees C led to 50 to 100-fold overproduction of EIImtl (5-10 mg/g of cell wet weight) relative to mannitol-induced levels in a wild-type (Mtl+) strain. Most of the overproduced protein was sequestered as an inactive form in inclusion bodies and cytoplasmic membranous structures. The protein could be extracted in an active form by rupturing the cells with lysozyme and sonication or with a passage through a French pressure cell and incubating the inclusion bodies and membranous structures with detergent (Lubrol PX or deoxycholate) in the presence of Q or S Sepharose ion-exchange resin for several hours. This procedure resulted in a 20- to 25-fold overproduction of active EIImtl compared with mannitol-induced wild-type levels.

Cloning, Molecular↗