RESUMO
The biocatalytic production of fuels and chemicals from plant biomass represents an attractive alternative to fossil fuel-based refineries. In this context, the mining and characterization of novel biocatalysts can promote disruptive innovation opportunities in the field of lignocellulose conversion and valorization. In the present work, we conducted the biochemical and structural characterization of two novel hydroxycinnamic acid catabolic enzymes, isolated from a lignin-degrading microbial consortium, a feruloyl-CoA synthetase, and a feruloyl-CoA hydratase-lyase, named LM-FCS2 and LM-FCHL2, respectively. Besides establishing the homology model structures for novel FCS and FCHL members with unique characteristics, the enzymes presented interesting biochemical features: LM-FCS2 showed stability in alkaline pHs and was able to convert a wide array of p-hydroxycinnamic acids to their respective CoA-thioesters, including sinapic acid; LM-FCHL2 efficiently converted feruloyl-CoA and p-coumaroyl-CoA into vanillin and 4-hydroxybenzaldehyde, respectively, and could produce vanillin directly from ferulic acid. The coupled reaction of LM-FCS2 and LM-FCHL2 produced vanillin, not only from commercial ferulic acid but also from a crude lignocellulosic hydrolysate. Collectively, this work illuminates the structure and function of two critical enzymes involved in converting ferulic acid into high-value molecules, thus providing valuable concepts applied to the development of plant biomass biorefineries. KEY POINTS: ⢠Comprehensive characterization of feruloyl-CoA synthetase from metagenomic origin. ⢠Novel low-resolution structures of hydroxycinnamate catabolic enzymes. ⢠Production of vanillin via enzymatic reaction using lignocellulosic hydrolysates.
Assuntos
Lignina , Metagenoma , Escherichia coli/genética , Hiperlipidemia Familiar Combinada , Lignina/metabolismo , SoloRESUMO
Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.
Assuntos
Bacillus licheniformis/enzimologia , Sacarose/análogos & derivados , Fosfatos Açúcares/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Sacarose/metabolismo , Difração de Raios XRESUMO
Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV-rNP). OROV-rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV-rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV-rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV-rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses.
Assuntos
Genoma Viral , Nucleoproteínas/química , Multimerização Proteica , RNA Viral/química , Vírus Simbu/química , Proteínas Virais/química , Humanos , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Vírus Simbu/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Gene expression in trypanosomes is controlled mostly by post-transcriptional pathways. Little is known about the components of mRNA nucleocytoplasmic export routes in these parasites. Comparative genomics has shown that the mRNA transport pathway is the least conserved pathway among eukaryotes. Nonetheless, we identified a RNA helicase (Hel45) that is conserved across eukaryotes and similar to shuttling proteins involved in mRNA export. We used in silico analysis to predict the structure of Trypanosoma cruzi Hel45, including the N-terminal domain and the C-terminal domain, and our findings suggest that this RNA helicase can form complexes with mRNA. Hel45 was present in both nucleus and cytoplasm. Electron microscopy showed that Hel45 is clustered close to the cytoplasmic side of nuclear pore complexes, and is also present in the nucleus where it is associated with peripheral compact chromatin. Deletion of a predicted Nuclear Export Signal motif led to the accumulation of Hel45ΔNES in the nucleus, indicating that Hel45 shuttles between the nucleus and the cytoplasm. This transport was dependent on active transcription but did not depend on the exportin Crm1. Knockdown of Mex67 in T. brucei caused the nuclear accumulation of the T. brucei ortholog of Hel45. Indeed, Hel45 is present in mRNA ribonucleoprotein complexes that are not associated with polysomes. It is still necessary to confirm the precise function of Hel45. However, this RNA helicase is associated with mRNA metabolism and its nucleocytoplasmic shuttling is dependent on an mRNA export route involving Mex67 receptor.
Assuntos
Proteínas de Protozoários/metabolismo , RNA Helicases/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Cultura Axênica , Domínio Catalítico , Núcleo Celular/enzimologia , Sequência Conservada , Citoplasma/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Poro Nuclear/enzimologia , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Helicases/química , RNA Helicases/genética , Transporte de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
UNLABELLED: Hemicellulose is an important part of the plant cell wall biomass, and is relevant to cellulosic ethanol technologies. ß-Mannosidases are enzymes capable of cleaving nonreducing residues of ß-d-mannose from ß-d-mannosides and hemicellulose mannose-containing polysaccharides, such as mannans and galactomannans. ß-Mannosidases are distributed between glycoside hydrolase (GH) families 1, 2, and 5, and only a handful of the enzymes have been structurally characterized to date. The only published X-ray structure of a GH family 2 mannosidase is that of the bacterial Bacteroides thetaiotaomicron enzyme. No structures of eukaryotic mannosidases of this family are currently available. To fill this gap, we set out to solve the structure of Trichoderma harzianum GH family 2 ß-mannosidase and to refine it to 1.9-Å resolution. Structural comparisons of the T. harzianum GH2 ß-mannosidase highlight similarities in its structural architecture with other members of GH family 2, reveal the molecular mechanism of ß-mannoside binding and recognition, and shed light on its putative galactomannan-binding site. DATABASE: Coordinates and observed structure factor amplitudes have been deposited with the Protein Data Bank (4CVU and 4UOJ). The T. harzianum ß-mannosidase 2A nucleotide sequence has GenBank accession number BankIt1712036 GeneMark.hmm KJ624918.
Assuntos
Proteínas Fúngicas/química , Trichoderma/enzimologia , beta-Manosidase/química , Proteínas de Bactérias/química , Domínio Catalítico , Cristalografia por Raios X , Proteínas Fúngicas/fisiologia , Galactose/análogos & derivados , Glicosilação , Mananas/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , beta-Manosidase/fisiologiaRESUMO
Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such as Aspergillus terreus are effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). The A. terreus GH12 endoglucanase was cloned and overexpressed in A. nidulans, purified and crystallized. A single crystal was obtained from a solution consisting of 2 M ammonium sulfate, 5%(v/v) 2-propanol. X-ray diffraction data were collected to a resolution of 1.85 Å using synchrotron radiation and a preliminary molecular-replacement solution was obtained in the trigonal space group P3(2)21. The unit-cell parameters were a = b = 103.24, c = 48.96 Å.
Assuntos
Aspergillus/enzimologia , Cristalografia por Raios X/métodos , Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Sequência de Bases , Cristalização , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Aiming to contribute toward the characterization of new, biotechnologically relevant cellulolytic enzymes, we report here the first crystal structure of the catalytic core domain of Cel7A (cellobiohydrolase I) from the filamentous fungus Trichoderma harzianum IOC 3844. Our structural studies and molecular dynamics simulations show that the flexibility of Tyr260, in comparison with Tyr247 from the homologous Trichoderma reesei Cel7A, is enhanced as a result of the short side-chains of adjacent Val216 and Ala384 residues and creates an additional gap at the side face of the catalytic tunnel. T. harzianum cellobiohydrolase I also has a shortened loop at the entrance of the cellulose-binding tunnel, which has been described to interact with the substrate in T. reesei Cel7A. These structural features might explain why T. harzianum Cel7A displays higher k(cat) and K(m) values, and lower product inhibition on both glucoside and lactoside substrates, compared with T. reesei Cel7A.
Assuntos
Celulose 1,4-beta-Celobiosidase/química , Simulação de Dinâmica Molecular , Trichoderma/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Ligação de Hidrogênio , Cinética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas de Protozoários/metabolismo , Transcrição Gênica , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Clonagem Molecular , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/citologia , Trypanosoma cruzi/fisiologiaRESUMO
Xylella fastidiosa was the first plant pathogen to have its complete genome sequence elucidated. Routine database analyses suggested that two enzymes essential for fatty acid synthesis were missing, one of these is the holo-acyl-carrier-protein synthase. However, here we demonstrate, using (13)C NMR spectroscopy, that X. fastidiosa is indeed able to synthesize fatty acids from acetate via an apparently conventional metabolic pathway. We further identify a gene product HetI, an alternative phosphopantetheinyl transferase, which we propose to fill the missing link. Homology modeling of HetI shows conservation of the Coenzyme A binding site suggesting it to be an active enzyme and reveals several interesting structural features when compared with the surfactin synthase-activating enzyme, on which the model was built. These include a simplified topology due to N- and C-terminal deletions and the observation of a novel serine ladder.
Assuntos
Ácidos Graxos/biossíntese , Perfilação da Expressão Gênica/métodos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Xylella/metabolismo , Acetatos/metabolismo , Sequência de Aminoácidos , Isótopos de Carbono , Simulação por Computador , Genoma Bacteriano , Dados de Sequência Molecular , Conformação Proteica , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Estatística como Assunto , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
The GumH enzyme from Xylella fastidiosa catalyzes the transfer reaction of a mannose from GDP-mannose to the carrier lipid cellobiose-pyrophosphate-polyprenol (Glc(2)-PP-Lip), an intermediary in the reaction for the synthesis of the exopolysaccharide (EPS) fastidian gum. The gumH gene was subcloned in the pMal-c2x vector, allowing the expression of the GumH-MBP fusion protein. Various attempts were made to obtain protein with the necessary degree of purity for crystallographic studies but the yield was very low. The gumH gene was then subcloned in the pET28a vector allowing the expression of the GumH enzyme in fusion with a histidine-rich peptide. The protein was purified and characterized. The three-dimensional structure of the X. fastidiosa GumH enzyme was modeled by threading studies. The model consists of N- and C-terminal domains similar in size and topology and separated by a deep cleft, which includes the EX(7)E motif that can be involved in the catalysis of GumH.