scholarly journals The Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region

1989 ◽  
Vol 260 (2) ◽  
pp. 601-604 ◽  
Author(s):  
M Leyh-Bouille ◽  
J Van Beeumen ◽  
S Renier-Pirlot ◽  
B Joris ◽  
M Nguyen-Distèche ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Active Site ◽  
Binding Protein ◽  
Terminal Region ◽  
High Homology ◽  
Penicillin Binding Protein ◽  
Serine Residue ◽  
Protein Active Site

The N-terminal region of the Streptomyces K15 DD-peptidase/penicillin-binding protein shows high homology with that of other penicillin-interactive proteins or domains. The active-site serine residue of the conserved tetrad Ser-Xaa-Xaa-Lys occurs at position 35. There is no indication for the presence of a signal peptide or an N-terminal hydrophobic sequence, suggesting that the Streptomyces K15 enzyme is probably anchored to the membrane by a C-terminal peptide segment.

scholarly journals Directly repeated insertion of 9-nucleotide sequence detected in penicillin-binding protein 2B gene of penicillin-resistant Streptococcus pneumoniae.

1996 ◽  
Vol 40 (5) ◽  
pp. 1257-1259 ◽  
Author(s):  
A Yamane ◽  
H Nakano ◽  
Y Asahi ◽  
K Ubukata ◽  
M Konno
Keyword(s):  
Streptococcus Pneumoniae ◽  
Nucleotide Sequence ◽  
Molecular Mechanism ◽  
Active Site ◽  
Binding Protein ◽  
Direct Repeat ◽  
Penicillin Binding Protein ◽  
Serine Residue ◽  
Class A ◽  
Class B

We investigated the molecular mechanism of 50 penicillin-resistant Streptococcus pneumoniae strains (penicillin: MIC, > or = 0.125 microgram/ml) having neither class A nor class B mutations in the penicillin-binding protein 2B gene (pbp2b). An analysis of the nucleotide sequences of the pbp2b genes from seven strains revealed an unique direct repeat of 9 nucleotides (TGGTATACT) between active-site serine (residue 385) and Ser-X-Asn (residues 442 to 444) motifs. The same insertion was detected in 13 strains.

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 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 Transcriptional Analysis of theStaphylococcus aureus Penicillin Binding Protein 2 Gene

1998 ◽  
Vol 180 (23) ◽  
pp. 6077-6081 ◽  
Author(s):  
Mariana G. Pinho ◽  
Herminia de Lencastre ◽  
Alexander Tomasz
Keyword(s):  
Bacillus Subtilis ◽  
Methicillin Resistance ◽  
Binding Protein ◽  
Primer Extension ◽  
Full Recovery ◽  
Transcriptional Analysis ◽  
High Homology ◽  
Penicillin Binding Protein ◽  
High Level

ABSTRACT Sequencing of the vicinity of the staphylococcal pbp2gene and transcriptional analysis by primer extension and promoter fusions were used to show that pbp2 is part of an operon that also includes a gene with high homology to prfA ofBacillus subtilis. Two distinct promoters were identified directing transcription of pbp2 either alone or together with prfA. It was recently reported that transposon inactivation of pbp2 causes a reduction in methicillin resistance, but complementation experiments were not fully successful. We now show that introduction of the intact pbp2 gene with its two newly identified promoters into the chromosome of the transposon mutant resulted in the full recovery of high-level methicillin resistance.

scholarly journals Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

1985 ◽  
Vol 164 (1) ◽  
pp. 456-460 ◽  
Author(s):  
R A Nicholas ◽  
J L Strominger ◽  
H Suzuki ◽  
Y Hirota
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Binding Protein ◽  
Penicillin Binding Protein

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 Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics

2020 ◽  
Vol 295 (21) ◽  
pp. 7529-7543
Author(s):  
Avinash Singh ◽  
Jonathan M. Turner ◽  
Joshua Tomberg ◽  
Alena Fedarovich ◽  
Magnus Unemo ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Protein Dynamics ◽  
Conformational Changes ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Transmitted Disease ◽  
Penicillin Binding Protein ◽  
Sexually Transmitted ◽  
Diminished Capacity

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the β3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the β3–β4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

Sign in / Sign up

Export Citation Format

Share Document