scholarly journals Catalytic mechanism of active-site serine β-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad

1994 ◽  
Vol 301 (2) ◽  
pp. 485-494 ◽  
Author(s):  
A Dubus ◽  
J M Wilkin ◽  
X Raquet ◽  
S Normark ◽  
J M Frère
Keyword(s):  
Hydroxy Group ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Peptidase Activity ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Class C

The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.

scholarly journals Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase

1992 ◽  
Vol 282 (2) ◽  
pp. 361-367 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
J M Wilkin ◽  
B Joris ◽  
R T Aplin ◽  
C Houssier ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Type Protein ◽  
Wild Type Protein ◽  
Tryptophan Residues

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

scholarly journals The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

1994 ◽  
Vol 301 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J M Wilkin ◽  
A Dubus ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

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 Mechanism of action of dd-peptidases: role of asparagine-161 in the Streptomyces R61 dd-peptidase

1993 ◽  
Vol 293 (1) ◽  
pp. 195-201 ◽  
Author(s):  
J M Wilkin ◽  
M Jamin ◽  
B Joris ◽  
J M Frere
Keyword(s):  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Peptide Substrate ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Wild Type Enzyme ◽  
Class A ◽  
First Case

The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis. The residue was successively replaced by serine and alanine. In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with the thiol-ester substrates and beta-lactams. However, the deacylation rates were decreased 10-30-fold with the substrates yielding benzoylglycyl and benzoylalanyl adducts. The Asn161Ala mutant was more generally affected, although the acylation rates with cefuroxime and cefotaxime remained similar to those observed with the wild-type enzyme. Surprisingly, the deacylation rates of the benzoylglycyl and benzoylalanyl adducts were very close to those observed with the wild-type enzyme. The results also indicate that the interaction with the peptide substrate and the transpeptidation reaction were more sensitive to the mutations than the other reactions studied. The results are discussed and compared with those obtained with the Asn-132 mutants of a class A beta-lactamase.

scholarly journals Insights into the role of lipoteichoic acids in Bacillus subtilis- a new function for MprF

2021 ◽  
Author(s):  
Aurelie Guyet ◽  
Amirah Alofi ◽  
Richard A Daniel
Keyword(s):  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Cellular Level ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
A Cell ◽  
Antimicrobial Peptide Resistance ◽  
Lipoteichoic Acids

In Bacillus subtilis, the cell is protected from the environment by a cell envelope, which comprises of layers of peptidoglycan that maintain the cell shape and anionic teichoic acids polymers whose biological function remains unclear. In B. subtilis, loss of all Class A Penicillin-Binding Proteins (aPBPs) which function in peptidoglycan synthesis is conditionally lethal. Here we show that this lethality is associated with an alteration of the lipoteichoic acids (LTA) and the accumulation of the major autolysin LytE in the cell wall. We provide the first evidence that the length and abundance of LTA acts to regulate the cellular level of LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF which acts to modulate LTA biosynthesis in B. subtilis and in the pathogen Staphylococcus aureus. This finding has implications for our understanding of antimicrobial peptide resistance (particularly daptomycin) in clinically relevant bacteria and MprF-associated virulence in pathogens, such as methicillin resistant S. aureus.

scholarly journals Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

1999 ◽  
Vol 43 (3) ◽  
pp. 543-548 ◽  
Author(s):  
Sonia Trépanier ◽  
James R. Knox ◽  
Natalie Clairoux ◽  
François Sanschagrin ◽  
Roger C. Levesque ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Enterobacter Cloacae ◽  
Acid Group ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Class C ◽  
Three Dimensional Models ◽  
Hydrolytic Mechanism

ABSTRACT Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the k cat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

scholarly journals Extended-Spectrum Cephalosporinase in Acinetobacter baumannii

2010 ◽  
Vol 54 (8) ◽  
pp. 3484-3488 ◽  
Author(s):  
José-Manuel Rodríguez-Martínez ◽  
Patrice Nordmann ◽  
Esthel Ronco ◽  
Laurent Poirel
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Isolate ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Extended Spectrum ◽  
Class C ◽  
High Level ◽  
Level Resistance

ABSTRACT An AmpC-type β-lactamase conferring high-level resistance to expanded-spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C β-lactamase (named ADC-33) possessed a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Ω-loop) compared to a reference AmpC cephalosporinase from A. baumannii. ADC-33 hydrolyzed ceftazidime, cefepime, and aztreonam at high levels, which allows the classification of this enzyme as an extended-spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property.

scholarly journals Imipenem as substrate and inhibitor of β-lactamases

1988 ◽  
Vol 253 (2) ◽  
pp. 323-328 ◽  
Author(s):  
J Monks ◽  
S G Waley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacillus Cereus ◽  
Reversible Reaction ◽  
Clinical Use ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A ◽  
Class C ◽  
Kinetics Of ◽  
Carbapenem Antibiotic

The interaction between imipenem, a carbapenem antibiotic, and two representative beta-lactamases has been studied. The first enzyme was beta-lactamase I, a class-A beta-lactamase from Bacillus cereus; imipenem behaved as a slow substrate (kcat. 6.7 min-1, Km 0.4 mM at 30 degrees C and at pH 7) that reacted by a branched pathway. There was transient formation of an altered species formed in a reversible reaction; this species was probably an acyl-enzyme in a slightly altered, but considerably more labile, conformation. The kinetics of the reaction were investigated by measuring both the concentration of the substrate and the activity of the enzyme, which fell and then rose again more slowly. The second enzyme was the chromosomal class-C beta-lactamase from Pseudomonas aeruginosa; imipenem was a substrate with a low kcat. (0.8 min-1) and a low Km (0.7 microM). Possible implications for the clinical use of imipenem are considered.

scholarly journals Synergy Between Beta-Lactams and Lipo-, Glyco-, and Lipoglycopeptides, Is Independent of the Seesaw Effect in Methicillin-Resistant Staphylococcus aureus

2021 ◽  
Vol 8 ◽  
Author(s):  
Rutan Zhang ◽  
Ismael A. Barreras Beltran ◽  
Nathaniel K. Ashford ◽  
Kelsi Penewit ◽  
Adam Waalkes ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Clinical Importance ◽  
Synergistic Activity ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Methicillin Resistant ◽  
Penicillin Binding Proteins ◽  
The Impact ◽  
Isogenic Strains

Methicillin-resistant S. aureus (MRSA) are resistant to beta-lactams, but synergistic activity between beta-lactams and glycopeptides/lipopeptides is common. Many have attributed this synergy to the beta-lactam-glycopeptide seesaw effect; however, this association has not been rigorously tested. The objective of this study was to determine whether the seesaw effect is necessary for synergy and to measure the impact of beta-lactam exposure on lipid metabolism. We selected for three isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin by serial passaging the MRSA strain N315. We used whole genome sequencing to identify genetic variants that emerged and tested for synergy between vancomycin, daptomycin, or dalbavancin in combination with 6 beta-lactams with variable affinity for staphylococcal penicillin binding proteins (PBPs), including nafcillin, meropenem, ceftriaxone, ceftaroline, cephalexin, and cefoxitin, using time-kills. We observed that the seesaw effect with each beta-lactam was variable and the emergence of the seesaw effect for a particular beta-lactam was not necessary for synergy between that beta-lactam and vancomycin, daptomycin, or dalbavancin. Synergy was more commonly observed with vancomycin and daptomycin based combinations than dalbavancin in time-kills. Among the beta-lactams, cefoxitin and nafcillin were the most likely to exhibit synergy using the concentrations tested, while cephalexin was the least likely to exhibit synergy. Synergy was more common among the resistant mutants than the parent strain. Interestingly N315-D1 and N315-DAL0.5 both had mutations in vraTSR and walKR despite their differences in the seesaw effect. Lipidomic analysis of all strains exposed to individual beta-lactams at subinhibitory concentrations suggested that in general, the abundance of cardiolipins (CLs) and most free fatty acids (FFAs) positively correlated with the presence of synergistic effects while abundance of phosphatidylglycerols (PGs) and lysylPGs mostly negatively correlated with synergistic effects. In conclusion, the beta-lactam-glycopeptide seesaw effect and beta-lactam-glycopeptide synergy are distinct phenomena. This suggests that the emergence of the seesaw effect may not have clinical importance in terms of predicting synergy. Further work is warranted to characterize strains that don’t exhibit beta-lactam synergy to identify which strains should be targeted with combination therapy and which ones cannot and to further investigate the potential role of CLs in mediating synergy.

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