RESUMO
The development and manufacture of high-quality starch are a new research focus in food science. Here, transglutaminase was used in the wet processing of glutinous rice flour to prepare customized sweet dumplings. Transglutaminase (0.2 %) lowered protein loss in wet processing and reduced the crystallinity and viscosity of glutinous rice flour. Moreover, it lowered the cracking and cooking loss of sweet dumplings after freeze-thaw cycles, and produced sweet dumplings with reduced hardness and viscosity, making them more suitable for people with swallowing difficulties. Additionally, in sweet dumplings with 0.2 % transglutaminase, the encapsulation of starch granules by the protein slowed down the digestion and reduced the final hydrolysis rate, which are beneficial for people with weight and glycemic control issues. In conclusion, this study contributes to the production of tasty, customized sweet dumplings.
Assuntos
Digestão , Farinha , Oryza , Amido , Transglutaminases , Oryza/química , Oryza/metabolismo , Transglutaminases/metabolismo , Transglutaminases/química , Farinha/análise , Amido/química , Amido/metabolismo , Manipulação de Alimentos , Humanos , Viscosidade , Culinária , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , BiocatáliseRESUMO
Phosphatidylethanolamine N-methyltransferase (PmtA) catalyzes the biosynthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE). Although PC is one of the major phospholipids constituting bilayer membranes in eukaryotes, certain bacterial species encode PmtA, a membrane-associated methyltransferase, to produce PC, which is correlated with cellular stress responses, adaptability to environmental changes, and symbiosis or virulence with eukaryotic hosts. Depending on the organism, multiple PmtAs may be required for producing monomethyl- and dimethyl-PE derivatives along with PC, whereas in organisms such as Rubellimicrobium thermophilum, a single enzyme is sufficient to direct all three methylation steps. In this study, we present the x-ray crystal structures of PmtA from R. thermophilum in complex with dimethyl-PE and S-adenosyl-l-homocysteine, as well as in its lipid-free form. Moreover, we demonstrate that the enzyme associates with the cellular membrane via electrostatic interactions facilitated by a group of critical basic residues and can successively methylate PE and its methylated derivatives, culminating in the production of PC.
Assuntos
Proteínas de Bactérias , Fosfatidilcolinas , Fosfatidiletanolamina N-Metiltransferase , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/biossíntese , Cristalografia por Raios X , Metilação , Membrana Celular/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosil-Homocisteína/química , Conformação Proteica , Ligação Proteica , Metiltransferases/metabolismo , Metiltransferases/química , Sequência de AminoácidosRESUMO
Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes. Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models. The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice. Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI).
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridioides difficile , Microscopia Crioeletrônica , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Animais , Clostridioides difficile/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Camundongos , Feminino , Infecções por Clostridium/imunologia , Infecções por Clostridium/microbiologia , Ligação Proteica , Humanos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Modelos MolecularesRESUMO
Inulin, a widely recognized prebiotic, has diverse applications across various industrial sectors. Although inulin is primarily produced through plant extraction, there is growing interest in enzymatic synthesis as an alternative. The enzymatic production of inulin from sucrose, which yields polymers with degrees of polymerization similar to those of plant-derived inulin, shows potential as a viable replacement for traditional extraction methods. In this study, an inulosucrase from Neobacillus bataviensis was identified, demonstrating a non-processive mechanism specifically tailored for synthesizing inulin with polymerization degrees ranging from 3 to approximately 40. The enzyme exhibited optimal activity at pH 6.5 and 55 °C, efficiently producing inulin with a yield of 50.6%. Ca2+ can improve the activity and thermostability of this enzyme. To enhance catalytic total activity, site-directed and truncated mutagenesis techniques were applied, resulting in the identification of a mutant, T149S, displaying a significant 57% increase in catalytic total activity. Molecular dynamics simulations unveiled that the heightened flexibility observed in three surface regions positively influenced enzymatic activity. This study not only contributes to the theoretical foundation for inulosucrase engineering but also presents a potential avenue for the production of inulin.
Assuntos
Hexosiltransferases , Inulina , Inulina/metabolismo , Hexosiltransferases/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/química , Simulação de Dinâmica Molecular , Concentração de Íons de Hidrogênio , Sacarose/metabolismo , Peso Molecular , Mutagênese Sítio-Dirigida , Bacillales/enzimologia , Bacillales/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Engenharia de Proteínas , Estabilidade Enzimática , Temperatura , Cálcio/metabolismoRESUMO
ß-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95â Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (ß/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of ß-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles.
Assuntos
beta-Glucosidase , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Cristalografia por Raios X , Caldicellulosiruptor/enzimologia , Modelos Moleculares , Conformação Proteica , Proteínas de Bactérias/química , Domínio Catalítico , Glucose/metabolismo , Sequência de AminoácidosRESUMO
Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations. Here, we attempt to replicate two recent studies reporting that deconstruction of polyethylene and poly(vinyl chloride) can be achieved using an insect hexamerin from Galleria mellonella (so-called "Ceres") or a bacterial catalase-peroxidase from Klebsiella sp., respectively. Reproducing previously described experiments, we do not observe any activity on plastics using multiple reaction conditions and multiple substrate types. Digging deeper into the discrepancies between the previous data and our observations, we show how and why the original experimental results may have been misinterpreted.
Assuntos
Biodegradação Ambiental , Klebsiella , Polietileno , Cloreto de Polivinila , Polietileno/metabolismo , Polietileno/química , Animais , Cloreto de Polivinila/química , Cloreto de Polivinila/metabolismo , Klebsiella/enzimologia , Klebsiella/metabolismo , Catalase/metabolismo , Catalase/química , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , BiocatáliseRESUMO
Overgrowth of Gardnerella vaginalis causes an imbalance in vaginal microecology. The pathogenicity of G. vaginalis is directly regulated by the cAMP receptor protein (CRP). In this study, we resolve the crystal structure of CRPGv at a resolution of 2.22 Å and find some significant differences from homologous proteins. The first 23 amino acids of CRPGv are inserted into the ligand binding pocket, creating a strong steric barrier to ligand entry that has not been seen previously in its homologues. In the absence of ligands, the two α helices used by CRPGv to bind oligonucleotide chains are exposed and can specifically bind TGTGA-N6-TCACA sequences. cAMP and other ligands of CRP homologs are not cofactors of CRPGv. There is no coding gene of the adenylate cyclase, and cAMP could not be identified in G. vaginalis by liquid chromatography tandem mass spectrometry. We speculate that CRPGv may achieve fine regulation through a conformational transformation different from that of its homologous proteins, and this conformational transformation is no longer dependent on small molecules, but may be aided by accessory proteins. CRPGv is the first discovered CRP that is not ligand-regulated, and its active conformation provides a structural basis for drug screening.
Assuntos
Gardnerella vaginalis , Ligantes , Gardnerella vaginalis/genética , Gardnerella vaginalis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/química , Cristalografia por Raios X , Sítios de Ligação , Modelos Moleculares , Sequência de Aminoácidos , Ligação Proteica , Conformação ProteicaRESUMO
Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T50 (incubation temperature at which 50% of the initial activity remains) 9 °C higher than that of wild-type MTG. In this study, we performed random mutations using D3C/G283C as a template and found several amino acid substitutions that contributed to the improvement of thermostability, and investigated a thermostable mutant (D3C/S101P/G157S/G250R/G283C) with three amino acid mutations in addition to the disulfide bond. The T50 of this mutant was 10 °C higher than that of the wild type, the optimal temperature for enzymatic reaction was increased to 65 °C compared to 50 °C for the wild type, and the catalytic efficiency (kcat/Km) at 37.0 °C was increased from 3.3 × 102 M-1 s-1 for the wild type to 5.9 × 102 M-1 s-1. X-ray crystallography of the D3C/G283C MTG showed no major structural differences against wild-type MTG. Structural differences were found that may contribute to thermostabilization and improve catalytic efficiency. KEY POINTS: ⢠Improved heat resistance is essential to broaden the application of MTG. ⢠The MTG mutant D3C/S101P/G157S/G250R/G283C showed improved thermostability. ⢠X-ray crystallography of the disulfide bridge mutant D3C/G283C MTG was elucidated.
Assuntos
Dissulfetos , Estabilidade Enzimática , Streptomyces , Transglutaminases , Streptomyces/enzimologia , Streptomyces/genética , Transglutaminases/genética , Transglutaminases/química , Transglutaminases/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Substituição de Aminoácidos , Mutagênese , Temperatura Alta , Temperatura , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , CinéticaRESUMO
Ribosomal protein SA (RPSA) plays multiple roles in cells, including ribosomal biogenesis and translation, cellular migration, and cytoskeleton reorganization. RPSA is crucial in the process of pathogen infection. Extensive research has examined RPSA's role in pathogen adhesion and invasion, but its broader functions, particularly its anti-infective capabilities, have garnered increasing attention in recent years. This dual role is closely related to its structural domains, which influence its localization and function. This review summarizes key research findings concerning the functional domains of RPSA and analyzes the relationship between its membrane localization and structural domains. Additionally, the functional implications of RPSA are categorized based on its different localizations during pathogen infection. Specifically, when RPSA is located on the cell surface, it promotes pathogen adhesion and invasion of host cells; conversely, when RPSA is located intracellularly, it exhibits anti-infective properties. Overall, RPSA shows a dual nature, both in facilitating pathogen invasion of the host and in possessing the ability to resist pathogen infection. This review comprehensively examines the dual role of RPSA in pathogen infection by analyzing its structural domains, localization, and interactions with cellular and pathogen molecules. Our aim is to update and deepen researchers' understanding of the various functions of RPSA during pathogen infection.
Assuntos
Proteínas Ribossômicas , Proteínas Ribossômicas/metabolismo , Humanos , Interações Hospedeiro-Patógeno , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , AnimaisRESUMO
The glycoside hydrolase family 3 (GH3) ß-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 ß-glucosidases. Two distinct configurations were identified in fungal GH3 ß-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in ß-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger ß-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric ß-glucosidases enzymes for industrial application.
Assuntos
Engenharia de Proteínas , Streptomyces , beta-Glucosidase , Streptomyces/enzimologia , Streptomyces/genética , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , beta-Glucosidase/química , Engenharia de Proteínas/métodos , Biotecnologia/métodos , Aspergillus niger/enzimologia , Aspergillus niger/genética , Domínios Proteicos , Aspergillus/enzimologia , Aspergillus/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Domínio Catalítico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/químicaRESUMO
To withstand their internal turgor pressure and external threats, most bacteria have a protective peptidoglycan (PG) cell wall. The growth of this PG polymer relies on autolysins, enzymes that create space within the structure. Despite extensive research, the regulatory mechanisms governing these PG-degrading enzymes remain poorly understood. Here, we unveil a novel and widespread control mechanism of lytic transglycosylases (LTs), a type of autolysin responsible for breaking down PG glycan chains. Specifically, we show that LD-crosslinks within the PG sacculus act as an inhibitor of LT activity. Moreover, we demonstrate that this regulation controls the release of immunogenic PG fragments and provides resistance against predatory LTs of both bacterial and viral origin. Our findings address a critical gap in understanding the physiological role of the LD-crosslinking mode in PG homeostasis, highlighting how bacteria can enhance their resilience against environmental threats, including phage attacks, through a single structural PG modification.
Assuntos
Parede Celular , N-Acetil-Muramil-L-Alanina Amidase , Peptidoglicano , Peptidoglicano/metabolismo , Parede Celular/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Escherichia coli/metabolismo , Glicosiltransferases/metabolismo , Bacillus subtilis/metabolismoRESUMO
Scarcity of structural and evolutionary information on protein complexes poses a challenge to deep learning-based structure modelling. We integrate experimental distance restraints obtained by crosslinking mass spectrometry (MS) into AlphaFold-Multimer, by extending AlphaLink to protein complexes. Integrating crosslinking MS data substantially improves modelling performance on challenging targets, by helping to identify interfaces, focusing sampling, and improving model selection. This extends to single crosslinks from whole-cell crosslinking MS, opening the possibility of whole-cell structural investigations driven by experimental data. We demonstrate this by revealing the molecular basis of iron homoeostasis in Bacillus subtilis.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Aprendizado Profundo , Espectrometria de Massas , Bacillus subtilis/metabolismo , Espectrometria de Massas/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Modelos Moleculares , Reagentes de Ligações Cruzadas/química , Ferro/metabolismo , Ferro/química , Conformação ProteicaRESUMO
Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp3)-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications.
Assuntos
Biocatálise , Engenharia de Proteínas , Streptomyces , Engenharia de Proteínas/métodos , Streptomyces/enzimologia , Streptomyces/genética , Ferroproteínas não Heme/química , Ferroproteínas não Heme/metabolismo , Ferroproteínas não Heme/genética , 4-Hidroxifenilpiruvato Dioxigenase/genética , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , 4-Hidroxifenilpiruvato Dioxigenase/química , Azidas/química , Azidas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
Rieske-type non-heme iron oxygenases (ROs) are an important family of non-heme iron enzymes. They catalyze a diverse range of transformations in secondary metabolite biosynthesis and xenobiotic bioremediation. ROs typically shuttle electrons from NAD(P)H to the oxygenase component via reductase component(s). This chapter describes our recent biochemical characterization of stachydrine demethylase Stc2 from Sinorhizobium meliloti. In this work, the eosin Y/sodium sulfite pair serves as the photoreduction system to replace the NAD(P)H-reductase system. We describe Stc2 protein purification and quality control details as well as a flow-chemistry to separate the photo-reduction half-reaction and the oxidation half-reaction. Our study demonstrates that the eosin Y/sodium sulfite photo-reduction pair is a NAD(P)H-reductase surrogate for Stc2-catalysis in a flow-chemistry setting. Experimental protocols used in this light-driven Stc2 catalysis are likely to be applicable as a photo-reduction system for other redox enzymes.
Assuntos
Oxirredução , Sinorhizobium meliloti , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/enzimologia , Sinorhizobium meliloti/metabolismo , Oxigenases/metabolismo , Oxigenases/genética , Oxigenases/química , Desmetilação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/químicaRESUMO
Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O2) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components.
Assuntos
Oxirredução , Oxigenases , Oxigenases/metabolismo , Oxigenases/química , Oxigenases/genética , Oxigenases/isolamento & purificação , Oxirredutases/metabolismo , Oxirredutases/química , Oxirredutases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Rhodococcus/enzimologia , Rhodococcus/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/químicaRESUMO
As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Here we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs.
Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Transporte Biológico , Microscopia Crioeletrônica , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Conformação ProteicaRESUMO
HerA is an ATP-dependent translocase that is widely distributed in archaea and some bacteria. It belongs to the HerA/FtsK translocase bacterial family, which is a subdivision of the RecA family. Currently, it is identified that HerA participates in the repair of DNA double-strand breaks (DSBs) or confers anti-phage defense by assembling other proteins into large complexes. In recent years, there has been a growing understanding of the bioinformatics, biochemistry, structure, and function of HerA subfamily members in both archaea and bacteria. This comprehensive review compares the structural disparities among diverse HerAs and elucidates their respective roles in specific life processes.
Assuntos
Proteínas de Bactérias , Evolução Molecular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Archaea/metabolismo , Archaea/genética , Reparo do DNA , Quebras de DNA de Cadeia Dupla , Bactérias/metabolismo , Modelos MolecularesRESUMO
Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.
Assuntos
Oxigenases , Pseudomonas fluorescens , Pseudomonas fluorescens/enzimologia , Oxigenases/metabolismo , Oxigenases/química , Dioxigenases/metabolismo , Dioxigenases/química , Dioxigenases/genética , Ensaios Enzimáticos/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de ElétronsRESUMO
The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins.
Assuntos
NAD , NAD/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Oxigenases/metabolismo , Oxigenases/química , Oxigenases/isolamento & purificação , Cristalografia por Raios X/métodos , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/isolamento & purificação , NADP/metabolismoRESUMO
Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.