scholarly journals PBP5 Complementation of a PBP3 Deficiency in Enterococcus hirae

2006 ◽  
Vol 188 (17) ◽  
pp. 6298-6307 ◽  
Author(s):  
S. Leimanis ◽  
N. Hoyez ◽  
S. Hubert ◽  
M. Laschet ◽  
Eric Sauvage ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Enterococcus Hirae ◽  
Penicillin Binding Proteins ◽  
Protein Protein Interaction ◽  
Interaction Sites ◽  
Functional Potential ◽  
The Stability ◽  
Protein Interaction Sites ◽  
Made In

ABSTRACT The low susceptibility of enterococci to β-lactams is due to the activity of the low-affinity penicillin-binding protein 5 (PBP5). One important feature of PBP5 is its ability to substitute for most, if not all, penicillin-binding proteins when they are inhibited. That substitution activity was analyzed in Enterococcus hirae SL2, a mutant whose pbp5 gene was interrupted by the nisRK genes and whose PBP3 synthesis was submitted to nisin induction. Noninduced SL2 cells were unable to divide except when plasmid-borne pbp5 genes were present, provided that the PBP5 active site was functional. Potential protein-protein interaction sites of the PBP5 N-terminal module were mutagenized by site-directed mutagenesis. The T167-L184 region (designated site D) appeared to be an essential intramolecular site needed for the stability of the protein. Mutations made in the two globular domains present in the N-terminal module indicated that they were needed for the suppletive activity. The P197-N209 segment (site E) in one of these domains seemed to be particularly important, as single and double mutations reduced or almost completely abolished, respectively, the action of PBP5.

scholarly journals Analysis of Escherichia coli RecA Interactions with LexA, λ CI, and UmuD by Site-Directed Mutagenesis of recA

2000 ◽  
Vol 182 (6) ◽  
pp. 1659-1670 ◽  
Author(s):  
Julie A. Mustard ◽  
John W. Little
Keyword(s):  
Escherichia Coli ◽  
Site Directed Mutagenesis ◽  
Early Event ◽  
Directed Mutagenesis ◽  
Alanine Scanning Mutagenesis ◽  
E Coli ◽  
Protein Protein Interaction ◽  
Interaction Sites ◽  
Lexa Repressor ◽  
Protein Interaction Sites

ABSTRACT An early event in the induction of the SOS system ofEscherichia coli is RecA-mediated cleavage of the LexA repressor. RecA acts indirectly as a coprotease to stimulate repressor self-cleavage, presumably by forming a complex with LexA. How complex formation leads to cleavage is not known. As an approach to this question, it would be desirable to identify the protein-protein interaction sites on each protein. It was previously proposed that LexA and other cleavable substrates, such as phage λ CI repressor andE. coli UmuD, bind to a cleft located between two RecA monomers in the crystal structure. To test this model, and to map the interface between RecA and its substrates, we carried out alanine-scanning mutagenesis of RecA. Twenty double mutations were made, and cells carrying them were characterized for RecA-dependent repair functions and for coprotease activity towards LexA, λ CI, and UmuD. One mutation in the cleft region had partial defects in cleavage of CI and (as expected from previous data) of UmuD. Two mutations in the cleft region conferred constitutive cleavage towards CI but not towards LexA or UmuD. By contrast, no mutations in the cleft region or elsewhere in RecA were found to specifically impair the cleavage of LexA. Our data are consistent with binding of CI and UmuD to the cleft between two RecA monomers but do not provide support for the model in which LexA binds in this cleft.

Crystallographic analysis and biochemical applications of a novel penicillin-binding protein/β-lactamase homologue from a metagenomic library

2014 ◽  
Vol 70 (9) ◽  
pp. 2455-2466 ◽  
Author(s):  
Tri Duc Ngo ◽  
Bum Han Ryu ◽  
Hansol Ju ◽  
Eun Jin Jang ◽  
Kyeong Kyu Kim ◽  
...  
Keyword(s):  
Metagenomic Library ◽  
Structural Features ◽  
Crystallographic Analysis ◽  
Penicillin Binding Protein ◽  
Hydrophobic Residues ◽  
Penicillin Binding Proteins ◽  
Hydrophobic Compounds ◽  
Nitrophenyl Phosphate ◽  
Biophysical Studies ◽  
The Stability

Interest in penicillin-binding proteins and β-lactamases (the PBP-βL family) is increasing owing to their biological and clinical significance. In this study, the crystal structure of Est-Y29, a metagenomic homologue of the PBP-βL family, was determined at 1.7 Å resolution. In addition, complex structures of Est-Y29 with 4-nitrophenyl phosphate (4NP) and with diethyl phosphonate (DEP) at 2.0 Å resolution were also elucidated. Structural analyses showed that Est-Y29 is composed of two domains: a β-lactamase fold and an insertion domain. A deep hydrophobic patch between these domains defines a wide active site, and a nucleophilic serine (Ser58) residue is located in a groove defined primarily by hydrophobic residues between the two domains. In addition, three hydrophobic motifs, which make up the substrate-binding site, allow this enzyme to hydrolyze a wide variety of hydrophobic compounds, including fish and olive oils. Furthermore, cross-linked Est-Y29 aggregates (CLEA-Est-Y29) significantly increase the stability of the enzyme as well as its potential for extensive reuse in various deactivating conditions. The structural features of Est-Y29, together with biochemical and biophysical studies, could provide a molecular basis for understanding the properties and regulatory mechanisms of the PBP-βL family and their potential for use in industrial biocatalysts.

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