RESUMO
Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.
Assuntos
Antibacterianos/química , Ceftazidima/química , Imipenem/química , Meropeném/química , Zinco/química , beta-Lactamases/química , Sequência de Aminoácidos , Antibacterianos/metabolismo , Domínio Catalítico , Cefepima/química , Cefepima/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Ceftazidima/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imipenem/metabolismo , Cinética , Meropeném/metabolismo , Modelos Moleculares , Piperacilina/química , Piperacilina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Zinco/metabolismo , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
The reaction between the antibiotic cefotaxime and the CTX-M-14 class A serine hydrolase is addressed from a theoretical point of view, by means of hybrid quantum mechanics/molecular mechanical (QM/MM) calculations, adopting a new approach that postulates that the residue Ser70 itself should play the role of the acid-base species required for the cefotaxime acylation. The proposed mechanism differs from earlier proposals existing in literature for other class A ß-lactamases. The results confirm the hypothesis, and show that the reaction should occur via a concerted mechanism in which the acylation of the lactam carbonyl carbon, protonation of the N7 lactam atom, and opening of the ß-lactam ring occurs simultaneously. Exploration of the potential energy surface shows three critical points, associated with reactants, transition state and product. The transition state is characterized by frequency, intrinsic reaction coordinate, atomic charge, and bond orders calculations. The calculated activation barrier is 20 kcal mol-1, and the reaction appears to be slightly endothermic by about 12 kcal mol-1. We conclude that this approach is feasible, and should be considered as an alternative mechanism or may exist in competition with others already published in the literature. This information should be useful for the design of novel antibiotics and ß-lactamase inhibitors. Graphical abstract Three-dimensional view of the potential energy surface of cefotaxime.
Assuntos
Cefotaxima/química , Simulação de Dinâmica Molecular , beta-Lactamases/química , Substituição de Aminoácidos , Mutação de Sentido Incorreto , Teoria Quântica , beta-Lactamases/genéticaRESUMO
Trace amounts of the widely used ß-lactam antibiotics (Atbs) in waste water may cause adverse effects on the ecosystems and contribute to the proliferation of antibiotic-resistant bacteria. On these grounds, kinetic and mechanistic aspects of photosensitized degradation of Ceftriaxone (Cft) and Cefotaxime (Ctx), have been studied in pure water by stationary and time-resolved techniques. Additionally, possible implications of these photoprocesses on the antimicrobial activity of the Atbs have also been investigated. Photoirradiation of aqueous solutions of Cft and Ctx produces the degradation of both Atbs in the presence of Riboflavin (vitamin B2), a well known pigment dissolved in natural aquatic systems. The process occurs through Type I and Type II mechanisms, with effective prevalence of the former. The participation of O2(-), OH and O2((1)Δg) is supported by experiments of oxygen consumption carried out in the presence of specific scavengers for such reactive oxygen species. Microbiological assays exhibit a parallelism between the rate of Cft and Ctx photodegradation and the loss of their bactericidal capacity on Staphylococcus aureus strains. Results contribute to both understanding kinetic and mechanism aspects of the degradation and predicting on natural decay of Atbs waste water-contaminants.
Assuntos
Antibacterianos/química , Cefotaxima/química , Ceftriaxona/química , Luz , Riboflavina/química , Antibacterianos/farmacologia , Cefotaxima/farmacologia , Ceftriaxona/farmacologia , Cinética , Oxirredução , Fotólise , Oxigênio Singlete/química , Espectrometria de Fluorescência , Staphylococcus aureus/efeitos dos fármacos , Superóxidos/química , Água/químicaRESUMO
A rapid procedure is reported for the synthesis of cefotaxime, by acylation of the 7-amino cephalosporanic acid with the 2-mercaptobenzothiazolyl thioester of (Z)-2-[2-aminothiazol-4-yl]-2-methoxyimino acetic acid (MAEM) that is a commercial reagent. The reaction was carried out at room temperature for 1 h, obtaining 95% yield. 2-Mercaptobenzothiazole was recovered as a side-product with a high purity and yield. The proposed method differentiates from those reported previously for a shorter time and very mild reaction condition, as well as for a ready for use reagent.
Assuntos
Cefotaxima/síntese química , Cefalosporinas/síntese química , Cefotaxima/química , Cefalosporinas/química , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Espectroscopia de Ressonância MagnéticaRESUMO
Cefotaxime sodium is a broad spectrum third generation antibiotic. It is obtained by reaction of 7-aminocephalosporanic acid (7-ACA) and S-(2-benzothiazolyl)2-amino-alpha-(methoxyimino)-4-thiazoleethanethio ate. 2-Mercaptobenzothiazole is a by-product of this reaction. A derivative spectrophotometric determination of cefotaxime is proposed for its determination in a reaction mixture in the presence of the related compounds from synthesis. With this method Beer's law is obeyed over a concentration range from 0.005 to 0.080 mg ml(-1) at 276.8 nm (r = 0.9995). This technique is accurate, precise (RSD = 0.4%), and has a sensitivity of 1.2% (differences in analytical response of 0.74 microg ml(-1) could be detected). Recovery experiments of cefotaxime from reaction mixtures include 100% for all assayed concentrations. For these reasons, this technique is found valid for the intended purposes.