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 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 The active sites of the β-lactamases of Streptomyces cacaoi and Streptomyces albus G.

1987 ◽  
Vol 244 (2) ◽  
pp. 427-432 ◽  
Author(s):  
F De Meester ◽  
B Joris ◽  
M V Lenzini ◽  
P Dehottay ◽  
T Erpicium ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Trypsin Digestion ◽  
Conserved Residues ◽  
Beta Lactamases ◽  
Class A ◽  
Denatured Proteins ◽  
Streptomyces Albus ◽  
High Degree

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.

scholarly journals Effect of side-chain amide thionation on turnover of β-lactam substrates by β-lactamases. Further evidence on the question of side-chain hydrogen-bonding in catalysis

1992 ◽  
Vol 286 (3) ◽  
pp. 857-862 ◽  
Author(s):  
R F Pratt ◽  
R Krishnaraj ◽  
H Xu
Keyword(s):  
Hydrogen Bond ◽  
Hydrogen Bonding ◽  
Essential Feature ◽  
Donor Site ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Beta Lactam ◽  
Hydrogen Bond Donor ◽  
Beta Lactamases ◽  
Beta Lactamase Ii

Two side-chain-thionated beta-lactams, a penicillin and a cephalosporin, have been prepared and found to be not significantly poorer as substrates of typical serine (classes A and C) beta-lactamases than are their oxo analogues. This result is interpreted to mean that any hydrogen-bonding site on these enzymes for the beta-lactam side-chain amide carbonyl group must be flexible and is more likely to be a passive rather than active or essential feature of the active site. Previously, data from crystal structures and site-directed mutagenesis had suggested that the side chain of Asn-132 of class-A beta-lactamases, a component of the conserved SDN loop, forms a hydrogen bond with the side-chain carbonyl of the beta-lactam substrate and may provide significant transition-state stabilization during catalysis. The thionocephalosporin was also equally as good as its oxo analogue as a substrate of the class-B beta-lactamase II of Bacillus cereus and not significantly less effective as an inhibitor of the Streptomyces R61 DD-peptidase; a tight hydrogen-bond donor site for the beta-lactam side-chain amide is apparently not present in these enzymes either.

scholarly journals Structural and kinetic studies on β-lactamase K1 from Klebsiella aerogenes

1986 ◽  
Vol 234 (2) ◽  
pp. 343-347 ◽  
Author(s):  
E L Emanuel ◽  
J Gagnon ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Klebsiella Aerogenes ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals A crystallographic study of the Toho-1 β-lactamase acylation mechanism

2014 ◽  
Vol 70 (a1) ◽  
pp. C1207-C1207
Author(s):  
Leighton Coates
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Ligand Complex ◽  
Transition State Analog ◽  
Hydrogen Bonding Interactions ◽  
Active Site Region ◽  
Neutron Crystallography

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

scholarly journals Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 985-994 ◽  
Author(s):  
Jin Kyun Kim ◽  
Cheol Lee ◽  
Seon Woo Lim ◽  
Jacob T. Andring ◽  
Aniruddha Adhikari ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Kinetic Parameters ◽  
Active Site ◽  
Systematic Approach ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Site Mutation ◽  
Functional Changes ◽  
Reaction Rate Constants

Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3 −) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (k cat and k cat/K m) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure–function relationships of many other biologically important enzymes.

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