RESUMO
Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.
Assuntos
Proteínas de Bactérias/metabolismo , Cafeína/química , Temperatura Baixa , Glucana 1,4-beta-Glucosidase/metabolismo , Sementes/química , Proteínas de Bactérias/química , Biocatálise , Cafeína/análise , Cobalto/química , Estabilidade Enzimática , Glucana 1,4-beta-Glucosidase/química , Paullinia/química , Xanthomonas/enzimologiaRESUMO
BACKGROUND: Enzymatic isomerization is a promising strategy to solve the problem of xylose fermentation and, consequently, to leverage the production of advanced biofuels and biochemicals. In a previous work, our research group discovered a new strain of Streptomyces with great biotechnological potential due to its ability to produce a broad arsenal of enzymes related to lignocellulose degradation. METHODS: We applied a multidisciplinary approach involving enzyme kinetics, biophysical methods, small angle X-ray scattering and X-ray crystallography to investigate two novel xylose isomerases, XylA1F1 and XylA2F1, from this strain. RESULTS: We showed that while XylA1F1 prefers to act at lower temperatures and relatively lower pH, XylA2F1 is extremely stable at higher temperatures and presents a higher turnover number. Structural analysis revealed that XylA1F1 exhibits unique properties in the active site not observed in classical XylAs from classes I and II nor in its ortholog XylA2F1. It encompasses the natural substitutions, M86A and T93K, that create an extra room for substrate accommodation and narrow the active-site entrance, respectively. Such modifications may contribute to the functional differentiation of these enzymes. CONCLUSIONS: We have characterized two novel xylose isomerases that display distinct functional behavior and harbor unprecedented amino-acid substitutions in the catalytic interface. GENERAL SIGNIFICANCE: Our findings contribute to a better understanding of the functional and structural aspects of xylose isomerases, which might be instrumental for the valorization of the hemicellulosic fraction of vegetal biomass.
Assuntos
Aldose-Cetose Isomerases/química , Streptomyces/enzimologia , Aldose-Cetose Isomerases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência , Streptomyces/química , Streptomyces/metabolismo , Especificidade por SubstratoRESUMO
Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites.
Assuntos
Antiprotozoários/farmacologia , Indóis/farmacologia , Leishmania major/enzimologia , Núcleosídeo-Difosfato Quinase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Calorimetria , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Leishmania major/efeitos dos fármacos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Testes de Sensibilidade Parasitária , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. Moreover, it is secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host-pathogen interactions. Here, we report the crystal structures of three mutants of the NDK from Leishmania major (LmNDK) that affects the stability of the hexameric biological assembly including P95S, Δ5Ct (lacking the last five residues) and the double mutant P100S/Δ5Ct. Although P95S and Δ5Ct variants conserve the hexameric structure of the wild-type protein, the double mutant becomes a dimer as shown by in solution studies. Free energy calculation of dimer-dimer interfaces and enzymatic assays indicate that P95S, Δ5Ct and P100S/Δ5Ct mutations progressively decrease the hexamer stability and enzyme activity. These results demonstrate that the mutated regions play a role in protein function through stabilizing the quaternary arrangement.
Assuntos
Leishmania major/enzimologia , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/ultraestrutura , Estrutura Quaternária de Proteína/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Interações Hospedeiro-Patógeno , Modelos MolecularesRESUMO
BACKGROUND: Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme that plays key roles in nucleotide recycling and homeostasis in trypanosomatids. It is also secreted by the intracellular parasite Leishmania to modulate the host response. These functions make NDK an attractive target for drug design and for studies aiming at a better understanding of the mechanisms mediating host-pathogen interactions. RESULTS: We report the crystal structure and biophysical characterization of the NDK from Leishmania braziliensis (LbNDK). The subunit consists of six α-helices along with a core of four ß-strands arranged in a ß2ß3ß1ß4 antiparallel topology order. In contrast to the NDK from L. major, the LbNDK C-terminal extension is partially unfolded. SAXS data showed that LbNDK forms hexamers in solution in the pH range from 7.0 to 4.0, a hydrodynamic behavior conserved in most eukaryotic NDKs. However, DSF assays show that acidification and alkalization decrease the hexamer stability. CONCLUSIONS: Our results support that LbNDK remains hexameric in pH conditions akin to that faced by this enzyme when secreted by Leishmania amastigotes in the parasitophorous vacuoles (pH 4.7 to 5.3). The unusual unfolded conformation of LbNDK C-terminus decreases the surface buried in the trimer interface exposing new regions that might be explored for the development of compounds designed to disturb enzyme oligomerization, which may impair the important nucleotide salvage pathway in these parasites.
Assuntos
Leishmania braziliensis/enzimologia , Núcleosídeo-Difosfato Quinase/química , Proteínas de Protozoários/química , Cristalografia por Raios X , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Leishmania braziliensis/química , Modelos Moleculares , Núcleosídeo-Difosfato Quinase/genética , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Proteínas de Protozoários/genética , Espalhamento a Baixo ÂnguloRESUMO
Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host-parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 angstrom resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 angstrom. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3(2)21.