scholarly journals Secretion by overexpression and purification of the water-soluble Streptomyces K15 dd-transpeptidase/penicillin-binding protein

1992 ◽  
Vol 288 (1) ◽  
pp. 87-91 ◽  
Author(s):  
P Palomeque-Messia ◽  
V Quittre ◽  
M Leyh-Bouille ◽  
M Nguyen-Distèche ◽  
C J L Gershater ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Binding Capacity ◽  
Binding Protein ◽  
Water Soluble ◽  
Appreciable Amount ◽  
Soluble Enzyme ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Membrane Bound

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity.

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

scholarly journals The Enterococcus hirae R40 penicillin-binding protein 5 and the methicillin-resistant Staphylococcus aureus penicillin-binding protein 2′ are similar

1991 ◽  
Vol 280 (2) ◽  
pp. 463-469 ◽  
Author(s):  
A el Kharroubi ◽  
P Jacques ◽  
G Piras ◽  
J Van Beeumen ◽  
J Coyette ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Water Soluble ◽  
Penicillin Binding Protein ◽  
Enterococcus Hirae ◽  
Methicillin Resistant ◽  
Class B ◽  
Membrane Bound ◽  
Polymerase Chain ◽  
Terminal Domains

The penicillin-resistant Enterococcus hirae R40 has a typical profile of membrane-bound penicillin-binding proteins (PBPs) except that the 71 kDa PBP5 of low penicillin affinity represents about 50% of all the PBPs present. Water-soluble tryptic-digest peptides were selectively produced from PBP5, their N-terminal regions were sequenced and synthetic oligonucleotides were used as primers to generate a 476 bp DNA fragment by polymerase chain reaction. On the basis of these data, the PBP5-encoding gene was cloned in Escherichia coli by using pBR322 as vector. The gene, included in a 7.1 kb insert, had the information for a 678-amino acid-residue protein. PBP5 shows similarity, in the primary structure, with the high-molecular-mass PBPs of class B. In particular, amino acid alignment of the enterococcal PBP5 and the methicillin-resistant staphylococcal PBP2′ generates scores that are 30, for the N-terminal domains, and 53, for the C-terminal domains, standard deviations above that expected for a run of 20 randomized pairs of proteins having the same amino acid compositions as the two proteins under consideration.

scholarly journals Active-site and membrane topology of the DD-peptidase/penicillin-binding protein no. 6 of Enterococcus hirae (Streptococcus faecium) A.T.C.C. 9790

1989 ◽  
Vol 262 (2) ◽  
pp. 457-462 ◽  
Author(s):  
A el Kharroubi ◽  
G Piras ◽  
P Jacques ◽  
I Szabo ◽  
J Van Beeumen ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Capacity ◽  
Binding Protein ◽  
Membrane Topology ◽  
Penicillin Binding Protein ◽  
Enterococcus Hirae ◽  
Serine Residue ◽  
Membrane Bound ◽  
High Degree ◽  
Terminal Appendage

The membrane-bound 43,000-Mr penicillin-binding protein no. 6 (PBP6) of Enterococcus hirae consists of a 30,000-Mr DD-peptidase/penicillin-binding domain and a approximately 130-residue C-terminal appendage. Removal of this appendage by trypsin proteolysis has no marked effect on the catalytic activity and penicillin-binding capacity of the PBP. Anchorage of the PBP in the membrane appears to be mediated by a short 15-20-residue stretch at the C-terminal end of the appendage. The sequence of the 50-residue N-terminal region of the PBP shows high degree of homology with the sequences of the corresponding regions of the PBPs5 of Escherichia coli and Bacillus subtilis. On this basis the active-site serine residue occurs at position 35 in the enterococcal PBP.

scholarly journals Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

1998 ◽  
Vol 180 (24) ◽  
pp. 6493-6502 ◽  
Author(s):  
Thomas Murray ◽  
David L. Popham ◽  
Christine B. Pearson ◽  
Arthur R. Hand ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Cell Elongation ◽  
Binding Protein ◽  
Divalent Cations ◽  
Cross Linking ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Spore Outgrowth ◽  
Partial Redundancy

ABSTRACT The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect inpbpA spore outgrowth have shown that (i) outgrowingpbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpAspores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpAspores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.

scholarly journals MppA, a Periplasmic Binding Protein Essential for Import of the Bacterial Cell Wall Peptidel-Alanyl-γ-d-Glutamyl-meso-Diaminopimelate

1998 ◽  
Vol 180 (5) ◽  
pp. 1215-1223 ◽  
Author(s):  
James T. Park ◽  
Debabrata Raychaudhuri ◽  
Hongshan Li ◽  
Staffan Normark ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Cell Wall ◽  
Binding Protein ◽  
Signal Transduction Pathway ◽  
Diaminopimelic Acid ◽  
Wild Type ◽  
Periplasmic Binding Protein ◽  
E Coli ◽  
New Gene ◽  
Membrane Bound ◽  
Oligopeptide Permease

ABSTRACT Mutants of a diaminopimelic acid (Dap)-requiring strain ofEscherichia coli were isolated which failed to grow on media in which Dap was replaced by the cell wall murein tripeptide,l-alanyl-γ-d-glutamyl-meso-diaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a new gene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, the periplasmic binding protein for Opp. A plasmid carrying the wild-typemppA gene allows the mutant to grow on tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity, indicating a defect in the oppoperon. An E. coli strain whose entire oppoperon was deleted but which carried the mppA locus was unable to grow on murein tripeptide unless it was provided withoppBCDF genes in trans. Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then transported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppA for non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys, which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, and perhaps the third OppA paralog revealed by the E. coli genome sequence may each bind a particular family of peptides but interact with common membrane-associated components for transport of their bound ligands into the cell. As to the physiological function of MppA, the possibility that it may be involved in signal transduction pathway(s) is discussed.

scholarly journals Primary and predicted secondary structures of the Actinomadura R39 extracellular dd-peptidase, a penicillin-binding protein (PBP) related to the Escherichia coli PBP4

1992 ◽  
Vol 282 (3) ◽  
pp. 781-788 ◽  
Author(s):  
B Granier ◽  
C Duez ◽  
S Lepage ◽  
S Englebert ◽  
J Dusart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Binding Capacity ◽  
Binding Protein ◽  
Secondary Structures ◽  
Streptomyces Lividans ◽  
Dimensional Structure ◽  
Peptidase Activity ◽  
Penicillin Binding Protein

As derived from gene cloning and sequencing, the 489-amino-acid DD-peptidase/penicillin-binding protein (PBP) produced by Actinomadura R39 has a primary structure very similar to that of the Escherichia coli PBP4 [Mottl, Terpstra & Keck (1991) FEMS Microbiol. Lett. 78, 213-220]. Hydrophobic-cluster analysis of the two proteins shows that, providing that a large 174-amino-acid stretch is excluded from the analysis, the bulk of the two polypeptide chains possesses homologues of the active-site motifs and secondary structures found in the class A beta-lactamase of Streptomyces albus G of known three-dimensional structure. The 174-amino-acid insert occurs at equivalent places in the two PBPs, between helices alpha 2 and alpha 3, away from the active site. Such an insert is unique among the penicilloyl serine transferases. It is proposed that the Actinomadura R39 PBP and E. coli PBP4 form a special class, class C, of low-Mr PBPs/DD-peptidases. A vector has been constructed and introduced by electrotransformation in the original Actinomadura R39 strain, allowing high-level expression and secretion of the DD-peptidase/PBP (250 mg.l-1). The gene encoding the desired protein is processed differently in Actinomadura R39 and Streptomyces lividans. Incorrect processing in Streptomyces lividans leads to a secreted protein which is inert in terms of DD-peptidase activity and penicillin-binding capacity.

scholarly journals A large displacement of the SXN motif of Cys115-modified penicillin-binding protein 5 from Escherichia coli

2005 ◽  
Vol 392 (1) ◽  
pp. 55-63 ◽  
Author(s):  
George Nicola ◽  
Alena Fedarovich ◽  
Robert A. Nicholas ◽  
Christopher Davies
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Major Change ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Penicillin Binding Proteins ◽  
Peptidoglycan Biosynthesis ◽  
Covalent Adduct

Penicillin-binding proteins (PBPs), which are the lethal targets of β-lactam antibiotics, catalyse the final stages of peptidoglycan biosynthesis of the bacterial cell wall. PBP 5 of Escherichia coli is a D-alanine CPase (carboxypeptidase) that has served as a useful model to elucidate the catalytic mechanism of low-molecular-mass PBPs. Previous studies have shown that modification of Cys115 with a variety of reagents results in a loss of CPase activity and a large decrease in the rate of deacylation of the penicilloyl–PBP 5 complex [Tamura, Imae and Strominger (1976) J. Biol. Chem. 251, 414–423; Curtis and Strominger (1978) J. Biol. Chem. 253, 2584–2588]. The crystal structure of wild-type PBP 5 in which Cys115 fortuitously had formed a covalent adduct with 2-mercaptoethanol was solved at 2.0 Å (0.2 nm) resolution, and these results provide a structural rationale for how thiol-directed reagents lower the rate of deacylation. When compared with the structure of the unmodified wild-type enzyme, a major change in the architecture of the active site is observed. The two largest differences are the disordering of a loop comprising residues 74–90 and a shift in residues 106–111, which results in the displacement of Ser110 of the SXN active-site motif. These results support the developing hypothesis that the SXN motif of PBP 5, and especially Ser110, is intimately involved in the catalytic mechanism of deacylation.

scholarly journals Affinity of Ceftobiprole for Penicillin-Binding Protein 2b in Streptococcus pneumoniae Strains with Various Susceptibilities to Penicillin

2010 ◽  
Vol 54 (10) ◽  
pp. 4510-4512 ◽  
Author(s):  
Todd A. Davies ◽  
Wenping He ◽  
Karen Bush ◽  
Robert K. Flamm
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Protein ◽  
Clinical Isolates ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
High Affinity

ABSTRACT Wild-type penicillin-binding protein (PBP) 2b from penicillin-susceptible Streptococcus pneumoniae had high affinity for ceftobiprole and penicillin (50% inhibitory concentrations [IC50s] of ≤0.15 μg/ml) but not ceftriaxone (IC50 of >8 μg/ml). In clinical isolates, ceftobiprole and PBP 2b affinities were reduced 15- to 30-fold with a Thr-446-Ala substitution and further still with an additional Ala-619-Gly PBP 2b substitution. Ceftobiprole remained active (MICs of ≤1 μg/ml) against all strains tested and behaved more like penicillin than ceftriaxone with respect to PBP 2b binding.

Sign in / Sign up

Export Citation Format

Share Document