RESUMO
A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/ß hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495µM and 0.24µM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.
Assuntos
Aspergillus/enzimologia , Epóxido Hidrolases/química , Proteínas Fúngicas/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Aspergillus/genética , Cromatografia de Afinidade , Dicroísmo Circular , Epóxido Hidrolases/genética , Epóxido Hidrolases/isolamento & purificação , Epóxido Hidrolases/metabolismo , Compostos de Epóxi/química , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Histidina/genética , Hidrólise , Dados de Sequência Molecular , Oligopeptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , EstereoisomerismoRESUMO
The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity.
Assuntos
5'-Nucleotidase/metabolismo , Xylella/enzimologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/isolamento & purificação , Biofilmes/crescimento & desenvolvimento , Coenzimas/metabolismo , Perfilação da Expressão Gênica , Metais/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Xylella/fisiologiaRESUMO
Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.
Assuntos
Proteínas de Bactérias/química , Escherichia coli , Lipoproteínas/química , Peptidoglicano/química , Redobramento de Proteína , Xylella , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Cromatografia em Gel , Lipoproteínas/biossíntese , Lipoproteínas/isolamento & purificação , Dados de Sequência Molecular , Peptidoglicano/biossíntese , Peptidoglicano/isolamento & purificação , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.