RESUMO
The cytoplasmatic biosynthesis of the stem peptide from the peptidoglycan in bacteria involves six steps, which have the role of three ATP-dependent Mur ligases that incorporate three consecutive amino acids to a substrate precursor. MurE is the last Mur ligase to incorporate a free amino acid. Although the structure of MurE from Mycobacterium tuberculosis (MtbMurE) was determined at 3.0 Å, the binding mode of meso-Diaminopimelate (m-DAP) and the effect of substrate absence is unknown. Herein, we show the structure of MurE from M. thermoresistibile (MthMurE) in complex with ADP and m-DAP at 1.4 Å resolution. The analysis of the structure indicates key conformational changes that the substrate UDP-MurNAc-L-Ala-D-Glu (UAG) and the free amino acid m-DAP cause on the MthMurE conformation. We observed several movements of domains or loop regions that displace their position in order to perform enzymatic catalysis. Since MthMurE has a high similarity to MtbMurE, this enzyme could also guide strategies for structure-based antimicrobial discovery to fight against tuberculosis or other mycobacterial infections.
Assuntos
Mycobacterium tuberculosis , Peptídeo Sintases , Peptídeo Sintases/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/metabolismo , AminoácidosRESUMO
BACKGROUND: Peptidoglycan (PG) is a key structural component of the bacterial cell wall and interruption of its biosynthesis is a validated target for antimicrobials. Of the enzymes involved in PG biosynthesis, D-alanyl,D-alanine ligase B (DdlB) is responsible for the condensation of two alanines, forming D-Ala-D-Ala, which is required for subsequent extracellular transpeptidase crosslinking of the mature peptidoglycan polymer. OBJECTIVE: We aimed at the biophysical characterization of recombinant Escherichia coli DdlB (EcDdlB), considering parameters of melting temperature (Tm), calorimetry and Van't Hoff enthalpy changes of denaturation ( ΔHUcal and ΔHUvH ), as well as characterization of elements of secondary structure at three different pHs. METHODS: DdlB was overexpressed in E. coli BL21 and purified by affinity chromatography. Thermal stability and structural characteristics of the purified enzyme were analyzed by circular dichroism (CD), differential scanning calorimetry and fluorescence spectroscopy. RESULTS: The stability of EcDdlB increased with proximity to its pI of 5.0, reaching the maximum at pH 5.4 with Tm and ΔHUvH U of 52.68 ºC and 484 kJ.mol-1, respectively. Deconvolutions of the CD spectra at 20 ºC showed a majority percentage of α-helix at pH 5.4 and 9.4, whereas for pH 7.4, an equal contribution of ß-structures and α-helices was calculated. Thermal denaturation process of EcDdlB proved to be irreversible with an increase in ß-structures that can contribute to the formation of protein aggregates. CONCLUSION: Such results will be useful for energy minimization of structural models aimed at virtual screening simulations, providing useful information in the search for drugs that inhibit peptidoglycan synthesis.
Assuntos
Escherichia coli , Peptidoglicano , Alanina , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Escherichia coli/genética , Ligases , Desnaturação Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Background: In view of the current low efficacy of bacterial infection treatment the common trend towards searching for antibiotic systems exhibiting synergistic action is well justified. Among carbapenem analogues a particularly interesting option is provided by combinations of clavulanic acid with meropenem, which have proven to be especially effective. Results: Determination of the minimal inhibitory concentration (MIC) along with the method based on flow cytometry constitutes an important tool in the identification of bacterial sensitivity to active substances. Within this study the inhibitory effect of doripenem, clavulanic acid and the doripenem-clavulanate acid system was analyzed in relation to such bacteria as Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Clostridium butyricum and Clostridium pasteurianum, Acinetobacter baumannii, Enterobacter aerogenes. The lowest MIC, amounting to 0.03 µg/mL, was observed for the doripenem-clavulanate acid system in the case of E. coli ATCC 25922. In turn, the lowest MIC for doripenem applied alone was recorded for K. pneumoniae ATCC 31488, for which it was 0.1 µg/mL. The strain which proved to be most resistant both to doripenem and the doripenem-clavulanate acid system, was A. baumannii, with MIC of 32 µg/mL (clinical isolate) and 16 µg/mL (reference strain). Cytometric analysis for P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 showed changes in cells following exposure to limiting concentrations of the active substance. Conclusions: Analysis of MIC supplies important information concerning microbial sensitivity to active substances, mainly in terms of limiting concentrations causing mortality or vitality of the tested species, which is essential when selecting appropriate antibiotic therapy.
Assuntos
Bactérias/efeitos dos fármacos , Ácidos Clavulânicos/farmacologia , Doripenem/farmacologia , Antibacterianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Salmonella/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Clostridium/efeitos dos fármacos , Interações Medicamentosas , Citometria de Fluxo , Klebsiella pneumoniae/efeitos dos fármacosRESUMO
The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018.