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Mutual influence of secondary and key drug-resistance mutations on catalytic properties and thermal stability of TEM-type ?-lactamases.

ABSTRACT: Highly mutable ?-lactamases are responsible for the ability of Gram-negative bacteria to resist ?-lactam antibiotics. Using site-directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM-type ?-lactamases, bearing the secondary substitution Q39K and key mutations related to the extended-spectrum (E104K, R164S) and inhibitor-resistant (M69V) ?-lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of ?-lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of ?-lactamase TEM-1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of ?-lactamase TEM-1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N-terminal ?-helix, towards the residues R244 and G236, located in the vicinity of the enzyme's catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of ?-lactamases can be used as a new potential target for inhibiting the enzyme.

SUBMITTER: Grigorenko V

PROVIDER: S-EPMC5757180 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Mutual influence of secondary and key drug-resistance mutations on catalytic properties and thermal stability of TEM-type β-lactamases.

Grigorenko Vitaly V Uporov Igor I Rubtsova Maya M Andreeva Irina I Shcherbinin Dmitrii D Veselovsky Alexander A Serova Oksana O Ulyashova Maria M Ishtubaev Igor I Egorov Alexey A

FEBS open bio 20171211 1

Highly mutable β-lactamases are responsible for the ability of Gram-negative bacteria to resist β-lactam antibiotics. Using site-directed mutagenesis technique, we have produced <i>in vitro</i> a number of recombinant analogs of naturally occurring TEM-type β-lactamases, bearing the secondary substitution Q39K and key mutations related to the extended-spectrum (E104K, R164S) and inhibitor-resistant (M69V) β-lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catal ...[more]

PMID: 29321962

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Project description:The most common mechanism of resistance to β-lactam antibiotics in Gram-negative bacteria is the production of β-lactamases that hydrolyze the drugs. Class A β-lactamases are serine active-site hydrolases that include the common TEM, CTX-M, and KPC enzymes. The TEM enzymes readily hydrolyze penicillins and older cephalosporins. Oxyimino-cephalosporins, such as cefotaxime and ceftazidime, however, are poor substrates for TEM-1 and were introduced, in part, to circumvent β-lactamase-mediated resistance. Nevertheless, the use of these antibiotics has lead to evolution of numerous variants of TEM with mutations that significantly increase the hydrolysis of the newer cephalosporins. The CTX-M enzymes emerged in the late 1980s and hydrolyze penicillins and older cephalosporins and derive their name from the ability to also hydrolyze cefotaxime. The CTX-M enzymes, however, do not efficiently hydrolyze ceftazidime. Variants of CTX-M enzymes, however, have evolved that exhibit increased hydrolysis of ceftazidime. Finally, the KPC enzyme emerged in the 1990s and is characterized by its broad specificity that includes penicillins, most cephalosporins, and carbapenems. The KPC enzyme, however, does not efficiently hydrolyze ceftazidime. As with the TEM and CTX-M enzymes, variants have recently evolved that extend the spectrum of KPC β-lactamase to include ceftazidime. This review discusses the structural and mechanistic basis for the expanded substrate specificity of each of these enzymes that result from natural mutations that confer oxyimino-cephalosporin resistance. For the TEM enzyme, extended-spectrum mutations act by establishing new interactions with the cephalosporin. These mutations increase the conformational heterogeneity of the active site to create sub-states that better accommodate the larger drugs. The mutations expanding the spectrum of CTX-M enzymes also affect the flexibility and conformation of the active site to accommodate ceftazidime. Although structural data are limited, extended-spectrum mutations in KPC may act by mediating new, direct interactions with substrate and/or altering conformations of the active site. In many cases, mutations that expand the substrate profile of these enzymes simultaneously decrease the thermodynamic stability. This leads to the emergence of additional global suppressor mutations that help correct the stability defects leading to increased protein expression and increased antibiotic resistance.

Dataset Information

Mutual influence of secondary and key drug-resistance mutations on catalytic properties and thermal stability of TEM-type ?-lactamases.

Publications

Mutual influence of secondary and key drug-resistance mutations on catalytic properties and thermal stability of TEM-type β-lactamases.

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