Catalytic Mechanism of theStreptomycesK15dd-Transpeptidase/Penicillin-Binding Protein Probed by Site-Directed Mutagenesis and Structural Analysis†,‡

Biochemistry ◽  
2003 ◽  
Vol 42 (10) ◽  
pp. 2895-2906 ◽  
Author(s):  
Noureddine Rhazi ◽  
Paulette Charlier ◽  
Dominique Dehareng ◽  
Danièle Engher ◽  
Marcel Vermeire ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Catalytic Mechanism ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein

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 Genetic Approach To Study the Relationship between Penicillin-Binding Protein 3 Mutations and Haemophilus influenzae β-Lactam Resistance by Using Site-Directed Mutagenesis and Gene Recombinants

2005 ◽  
Vol 49 (7) ◽  
pp. 2834-2839 ◽  
Author(s):  
Yumi Osaki ◽  
Yumiko Sanbongi ◽  
Midori Ishikawa ◽  
Hiroshi Kataoka ◽  
Takahisa Suzuki ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Binding Protein ◽  
Antibiotic Sensitivity ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Recombinant Strains ◽  
Amino Acid Mutations ◽  
The Relationship

ABSTRACT To clarify the relationship between mutations commonly found for penicillin-binding protein 3 (PBP 3) of β-lactamase-nonproducing ampicillin-resistant (BLNAR) Haemophilus influenzae isolates and β-lactam resistance, single and multiple amino acid mutations at positions 377, 385, 389, 517, and 526 were introduced into PBP 3 of a β-lactam-susceptible Rd strain by site-directed mutagenesis. Twelve isogenic recombinant strains were challenged with nine β-lactam antibiotics. Replacement of the asparagine at position 526 with lysine (N526K) increased the resistance to imipenem eightfold and increased the resistance to various cephalosporins two- to eightfold. Substitution of threonine for serine at position 385 (S385T) and/or substitution of phenylalanine for leucine at position 389 (L389F), in addition to the N526K mutation, led to two- to fourfold additional increases in cephalosporin resistance. An isoleucine-to-methionine substitution at position 377 did not change the antibiotic sensitivity of any of the recombinant strains also carrying other PBP 3 mutations tested. Thirty-six clinical isolates carrying a PBP 3 gene (ftsI) with the S385T, L389F, R517H, and/or N526K mutation were chosen from among 279 clinical isolates collected in Japan, and the isolates were grouped into six classes on the basis of the patterns of the four mutations in PBP 3. Rd recombinants were made with each of the ftsI genes. The levels of resistance to β-lactams varied between recombinants of different classes but were comparable for those of the same class. The levels of resistance to cephalosporins of these recombinants were similar to those of the parent clinical isolates, while those to ampicillin and carbapenems were lower. These results indicate that resistance to β-lactams, at least to cephalosporins, depends in large part on the PBP 3 mutations R517H, N526K, S385T, and L389F.

scholarly journals Site-directed mutagenesis of penicillin-binding protein 3 ofEscherichia coli: Role of Val-545

1994 ◽  
Vol 121 (2) ◽  
pp. 251-256 ◽  
Author(s):  
Juan Ayala ◽  
Colette Goffin ◽  
Martine Nguyen-Distèche ◽  
Jean-Marie Ghuysen
Keyword(s):  
Binding Protein ◽  
Ofescherichia Coli ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein

β-Amyrin biosynthesis: catalytic mechanism and substrate recognition

2017 ◽  
Vol 15 (14) ◽  
pp. 2869-2891 ◽  
Author(s):  
Tsutomu Hoshino
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The Past ◽  
Substrate Analog ◽  
Analog Experiments

In the past five years, there have been remarkable advances in the study of β-amyrin synthase. This review outlines the catalytic mechanism and substrate recognition in β-amyrin biosynthesis, which have been attained by the site-directed mutagenesis and substrate analog experiments.

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