RESUMO
Understanding the evolution of metallo-ß-lactamases (MBLs) is fundamental to deciphering the mechanistic basis of resistance to carbapenems in pathogenic and opportunistic bacteria. Presently, these MBL-producing pathogens are linked to high rates of morbidity and mortality worldwide. However, the study of the biochemical and biophysical features of MBLs in vitro provides an incomplete picture of their evolutionary potential, since this limited and artificial environment disregards the physiological context where evolution and selection take place. Herein, we describe recent efforts aimed to address the evolutionary traits acquired by different clinical variants of MBLs in conditions mimicking their native environment (the bacterial periplasm) and considering whether they are soluble or membrane-bound proteins. This includes addressing the metal content of MBLs within the cell under zinc starvation conditions and the context provided by different bacterial hosts that result in particular resistance phenotypes. Our analysis highlights recent progress bridging the gap between in vitro and in-cell studies.
Assuntos
Bactérias , Periplasma , beta-Lactamases , Antibacterianos/química , Bactérias/enzimologia , Bactérias/metabolismo , Carbapenêmicos , Periplasma/enzimologia , Periplasma/metabolismo , beta-Lactamases/químicaRESUMO
Enzymatic prospection indicated that L-asparaginase from Erwinia carotovora (ECAR-LANS) posses low glutaminase activity and much effort has been made to produce therapeutic ECAR-LANS. However, its low stability precludes its use in therapy. Herein, biochemical and biophysical assays provided data highlighting the influence of solubilization and storage into ECAR-LANS structure, stability, and activity. Moreover, innovations in recombinant expression and purification guaranteed the purification of functional tetramers. According to solubilization condition, the L-asparaginase activity and temperature of melting ranged up to 25-32%, respectively. CD spectra indicate the tendency of ECAR-LANS to instability and the influence of ß-structures in activity. These results provide relevant information to guide formulations with prolonged action in the bloodstream.
Assuntos
Asparaginase/metabolismo , Pectobacterium carotovorum/enzimologia , Citoplasma/enzimologia , Estabilidade Enzimática , Fluorescência , Periplasma/enzimologiaRESUMO
Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2' ]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90â Å resolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84â Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.
Assuntos
Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/isolamento & purificação , Periplasma/enzimologia , Xanthomonadaceae/enzimologia , Cristalização , Cristalografia por Raios X , Dipeptidil Peptidase 4/metabolismo , Cinética , Conformação Proteica , Xanthomonadaceae/classificaçãoRESUMO
Metallo-beta-lactamases (MBLs) are broad-spectrum, Zn(II)-dependent lactamases able to confer resistance to virtually every ß-lactam antibiotic currently available. The large diversity of active-site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3 that is expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica This MBL is atypical, since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here, we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active-site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono- and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins, and carbapenems. In silico docking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast to Ser221, which otherwise is conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design and also posit that GOB enzymes have alternative functions.
Assuntos
Farmacorresistência Bacteriana Múltipla , Flavobacteriaceae/enzimologia , Glutamina/química , Histidina/química , Zinco/química , beta-Lactamases/química , Antibacterianos/química , Antibacterianos/metabolismo , Carbapenêmicos/química , Carbapenêmicos/metabolismo , Domínio Catalítico , Cátions Bivalentes , Cefalosporinas/química , Cefalosporinas/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Flavobacteriaceae/química , Expressão Gênica , Glutamina/metabolismo , Histidina/metabolismo , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Penicilinas/química , Penicilinas/metabolismo , Periplasma/química , Periplasma/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
ß-Fructofuranosidases or invertases (EC 3.2.1.26) are enzymes that are widely used in the food industry, where fructose is preferred over sucrose, because it is sweeter and does not crystallize easily. Since Aspergillus niger GH1, an xerophilic fungus from the Mexican semi-desert, has been reported to be an invertase producer, and because of the need for new enzymes with biotechnological applications, in this work, we describe the gene and amino acid sequence of the invertase from A. niger GH1, and the use of a synthetic gene to produce the enzyme in the methylotrophic yeast Pichia pastoris. In addition, the produced invertase was characterized biochemically. The sequence of the invertase gene had a length of 1770 bp without introns, encodes a protein of 589 amino acids, and presented an identity of 93% and 97% with invertases from Aspergillus kawachi IFO 4308 and A. niger B60, respectively. A 4.2 L culture with the constructed recombinant P. pastoris strain showed an extracellular and periplasmic invertase production at 72 h induction of 498 and 3776 invertase units (U), respectively, which corresponds to 1018 U/L of culture medium. The invertase produced had an optimum pH of 5.0, optimum temperature of 60 °C, and specific activity of 3389 U/mg protein, and after storage for 96 h at 4 °C showed 93.7% of its activity. This invertase could be suitable for producing inverted sugar used in the food industry.
Assuntos
Aspergillus niger/genética , Proteínas Fúngicas/genética , beta-Frutofuranosidase/genética , Sequência de Aminoácidos , Aspergillus niger/enzimologia , Sequência de Bases , Sistema Livre de Células , Clonagem Molecular , Líquido Extracelular/enzimologia , Frutose/biossíntese , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Genes Sintéticos , Glucose/biossíntese , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Periplasma/enzimologia , Pichia , Estabilidade Proteica , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência , Sacarose/metabolismo , Temperatura , beta-Frutofuranosidase/isolamento & purificação , beta-Frutofuranosidase/metabolismoRESUMO
Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-ß-lactamases (MßLs), enzymes able to hydrolyze most ß-lactam antibiotics. SPM-1 is an MßL produced only by P. aeruginosa, while other MßLs are found in different bacteria. Despite similar active sites, the resistance profile of MßLs towards ß-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MßLs in that it contains "atypical" second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards ß-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.
Assuntos
Mutação , Periplasma/enzimologia , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/química , Animais , Domínio Catalítico , Estabilidade Enzimática , Periplasma/genética , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa/genética , Coelhos , Especificidade por Substrato , Resistência beta-Lactâmica/fisiologia , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
In this work the periplasmic enzymatic complex glucose-fructose oxidoreductase (GFOR)/glucono-δ-lactonase (GL) of permeabilized free or immobilized cells of Zymomonas mobilis was evaluated for the bioconversion of mixtures of fructose and different aldoses into organic acids. For all tested pairs of substrates with permeabilized free-cells, the best enzymatic activities were obtained in reactions with pH around 6.4 and temperatures ranging from 39 to 45 °C. Decreasing enzyme/substrate affinities were observed when fructose was in the mixture with glucose, maltose, galactose, and lactose, in this order. In bioconversion runs with 0.7 mol l(-1) of fructose and with aldose, with permeabilized free-cells of Z. mobilis, maximal concentrations of the respective aldonic acids of 0.64, 0.57, 0.51, and 0.51 mol l(-1) were achieved, with conversion yields of 95, 88, 78, and 78 %, respectively. Due to the important applications of lactobionic acid, the formation of this substance by the enzymatic GFOR/GL complex in Ca-alginate-immobilized cells was assessed. The highest GFOR/GL activities were found at pH 7.0-8.0 and temperatures of 47-50 °C. However, when a 24 h bioconversion run was carried out, it was observed that a combination of pH 6.4 and temperature of 47 °C led to the best results. In this case, despite the fact that Ca-alginate acts as a barrier for the diffusion of substrates and products, maximal lactobionic acid concentration, conversion yields and specific productivity similar to those obtained with permeabilized free-cells were achieved.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Oxirredutases/metabolismo , Zymomonas/enzimologia , Alginatos , Células Imobilizadas/enzimologia , Dissacarídeos/biossíntese , Frutose/metabolismo , Galactose/metabolismo , Ácido Glucurônico , Ácidos Hexurônicos , Maltose/metabolismo , Periplasma/enzimologiaRESUMO
Chemolithoautotrophic acidophilic bacteria, which belong to the genus Leptospirillum, can only grow with Fe(II) as electron donor and oxygen as an electron acceptor. Members of this genus play an important role in bioleaching sulfide ores. We used nearly complete genome sequences of Leptospirillum ferrooxidans (group I), Leptospirillum rubarum, Leptospirillum '5-way CG' (group II) and Leptospirillum ferrodiazotrophum (group III) to identify cytochromes that are likely involved in electron transfer chain(s). The results show the presence of genes encoding a number of c-type cytochromes (18-20 genes were identified in each species), as well as bd and cbb3 oxidases. Genes encoding cbb3 oxidase are clustered, with predicted genes involved in cbb3 maturation proteins. Duplication of cbb3 encoding genes (ccoNO) was detected in all four genomes. Interestingly, these micro-organisms also contain genes that potentially encode bc1 and b6f-like complexes organized into two putative operon structures. To date, the Leptospirillum genus includes the only organisms reported to have genes coding for two different bc complexes. This study provides detailed insights into the components of electron transfer chains of Leptospirillum spp., revealing their conservation among leptospirilla groups and suggesting that there may be a single common pathway for electron transport between Fe(II) and oxygen.
Assuntos
Bactérias/genética , Grupo dos Citocromos c/genética , Genoma Bacteriano , Bactérias/classificação , Bactérias/enzimologia , Hibridização Genômica Comparativa , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Compostos Férricos/metabolismo , Duplicação Gênica , Óperon , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Periplasma/enzimologia , FilogeniaRESUMO
Gluconacetobacter diazotrophicus utilizes plant sucrose with a constitutively expressed levansucrase (LsdA), producing extracellular levan, which may be degraded under energetically unfavored conditions. Reverse transcriptase-PCR analysis revealed that lsdA and the downstream exolevanase gene (lsdB) form an operon. lsdB transcription was induced during growth with low fructose concentrations (0.44 to 33 mM) and repressed by glucose. Transport of LsdB to the periplasm involved N-terminal signal peptide cleavage. Type II secretion mutants failed to transfer LsdB across the outer membrane, impeding levan hydrolysis.
Assuntos
Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Gluconacetobacter/enzimologia , Gluconacetobacter/genética , Glicosídeo Hidrolases/biossíntese , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Frutanos/metabolismo , Frutose/metabolismo , Ordem dos Genes , Glucose/metabolismo , Glicosídeo Hidrolases/genética , Dados de Sequência Molecular , Óperon , Periplasma/enzimologia , Sinais Direcionadores de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]-linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N(2) or NH(3)). Its synthesis was stimulated by conditions of high energetic demand (i.e., N(2)-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N(2)-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)-linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.