Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A .beta.-lactamases

Biochemistry ◽  
1992 ◽  
Vol 31 (2) ◽  
pp. 430-437 ◽  
Author(s):  
Hiroyuki Adachi ◽  
Masaji Ishiguro ◽  
Seiichi Imajoh ◽  
Takahisa Ohta ◽  
Hiroshi Matsuzawa
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Active Site Residues ◽  
Beta Lactamases ◽  
Class A

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 Amino acid sequence of the penicillin-binding protein/dd-peptidase of Streptomyces K15. Predicted secondary structures of the low Mr penicillin-binding proteins of class A

1991 ◽  
Vol 279 (1) ◽  
pp. 223-230 ◽  
Author(s):  
P Palomeque-Messia ◽  
S Englebert ◽  
M Leyh-Bouille ◽  
M Nguyen-Distèche ◽  
C Duez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Active Sites ◽  
Binding Protein ◽  
Secondary Structures ◽  
Peptide Sequence ◽  
Penicillin Binding Protein ◽  
Signal Peptide Sequence ◽  
Beta Lactamases ◽  
Class A

The low-Mr penicillin-binding protein (PBP)/DD-transpeptidase of Streptomyces K15 is synthesized in the form of a 291-amino acid-residue precursor possessing a cleavable 29-amino acid-residue signal peptide. Sequence-similarity searches and hydrophobic-cluster analysis show that the Streptomyces K15 enzyme, the Escherichia coli PBPs/DD-carboxy-peptidases 5 and 6, the Bacillus subtilis PBP/DD-carboxypeptidase 5 and the spoIIA product (a putative PBP involved in the sporulation of B. subtilis) are structurally related and form a distinct class A of low-Mr PBPs/DD-peptidases. The distribution of the hydrophobic clusters along the amino acid sequences also shows that the Streptomyces K15 PBP, and by extension the other PBPs of class A, have similarity in the polypeptide folding, with the beta-lactamases of class A, with as reference the Streptomyces albus G and Staphylococcus aureus beta-lactamases of known three-dimensional structure. This comparison allows one to predict most of the secondary structures in the PBPs and the amino acid motifs that define the enzyme active sites.

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 Streptomyces albus G serine β-lactamase. Probing of the catalytic mechanism via molecular modelling of mutant enzymes

1992 ◽  
Vol 282 (1) ◽  
pp. 189-195 ◽  
Author(s):  
J Lamotte-Brasseur ◽  
F Jacob-Dubuisson ◽  
G Dive ◽  
J M Frère ◽  
J M Ghuysen
Keyword(s):  
Hydrogen Bonding ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Network Configuration ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Streptomyces Albus ◽  
Hydrogen Bonding Network

In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase [Lamotte-Brasseur, Dive, Dideberg, Charlier, Frère & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.

scholarly journals Identification and overexpression in Escherichia coli of a Mycobacterium leprae gene, pon1, encoding a high-molecular-mass class A penicillin-binding protein, PBP1.

1996 ◽  
Vol 178 (6) ◽  
pp. 1707-1711 ◽  
Author(s):  
J Basu ◽  
S Mahapatra ◽  
M Kundu ◽  
S Mukhopadhyay ◽  
M Nguyen-Distèche ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Binding Protein ◽  
Mycobacterium Leprae ◽  
High Molecular Mass ◽  
Penicillin Binding Protein ◽  
Class A

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

1992 ◽  
Vol 174 (10) ◽  
pp. 3261-3269 ◽  
Author(s):  
H Mottl ◽  
P Nieland ◽  
G de Kort ◽  
J J Wierenga ◽  
W Keck
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Additional Domain

scholarly journals The ponA Gene of Enterococcus faecalis JH2-2 Codes for a Low-Affinity Class A Penicillin-Binding Protein

2004 ◽  
Vol 186 (13) ◽  
pp. 4412-4416 ◽  
Author(s):  
Colette Duez ◽  
Séverine Hallut ◽  
Noureddine Rhazi ◽  
Séverine Hubert ◽  
Ana Amoroso ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterococcus Faecalis ◽  
Binding Proteins ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Lipid Ii

ABSTRACT A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a dd- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind β-lactams, but with k 2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.

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