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1.
Nat Commun ; 11(1): 4808, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968058

RESUMO

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.


Assuntos
Simulação por Computador , Evolução Molecular Direcionada/métodos , Enzimas/química , Evolução Química , Liases/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Enzimas/genética , Enzimas/metabolismo , Cinética , Liases/genética , Liases/metabolismo , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Engenharia de Proteínas
2.
Nat Commun ; 11(1): 4292, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855421

RESUMO

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.


Assuntos
Bioquímica/métodos , Butanóis/metabolismo , Enzimas/metabolismo , Acetolactato Sintase/química , Acetolactato Sintase/metabolismo , Trifosfato de Adenosina , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Bioquímica/instrumentação , Reatores Biológicos , Sistema Livre de Células , Evolução Molecular Direcionada , Enzimas/química , Enzimas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Temperatura , Termodinâmica
3.
Methods Mol Biol ; 2203: 187-204, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833213

RESUMO

Biotin-based proximity labeling circumvents major pitfalls of classical biochemical approaches to identify protein-protein interactions. It consists of enzyme-catalyzed biotin tags ubiquitously apposed on proteins located in close proximity of the labeling enzyme, followed by affinity purification and identification of biotinylated proteins by mass spectrometry. Here we outline the methods by which the molecular microenvironment of the coronavirus replicase/transcriptase complex (RTC), i.e., proteins located within a close perimeter of the RTC, can be determined by different proximity labeling approaches using BirAR118G (BioID), TurboID, and APEX2. These factors represent a molecular signature of coronavirus RTCs and likely contribute to the viral life cycle, thereby constituting attractive targets for the development of antiviral intervention strategies.


Assuntos
Coronavirus/patogenicidade , Enzimas/genética , Interações Hospedeiro-Patógeno/fisiologia , Proteômica/métodos , Proteínas Virais/metabolismo , Animais , Ascorbato Peroxidases/genética , Biotinilação , Carbono-Nitrogênio Ligases/genética , Linhagem Celular , Coronavirus/genética , Enzimas/metabolismo , Proteínas de Escherichia coli/genética , Imunofluorescência , Microrganismos Geneticamente Modificados , Proteínas Repressoras/genética , Proteínas Virais/química , Proteínas Virais/genética
4.
Nucleic Acids Res ; 48(W1): W110-W115, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32406917

RESUMO

The CUPP platform includes a web server for functional annotation and sub-grouping of carbohydrate active enzymes (CAZymes) based on a novel peptide-based similarity assessment algorithm, i.e. protein grouping according to Conserved Unique Peptide Patterns (CUPP). This online platform is open to all users and there is no login requirement. The web server allows the user to perform genome-based annotation of carbohydrate active enzymes to CAZy families, CAZy subfamilies, CUPP groups and EC numbers (function) via assessment of peptide-motifs by CUPP. The web server is intended for functional annotation assessment of the CAZy inventory of prokaryotic and eukaryotic organisms from genomic DNA (up to 30MB compressed) or directly from amino acid sequences (up to 10MB compressed). The custom query sequences are assessed using the CUPP annotation algorithm, and the outcome is displayed in interactive summary result pages of CAZymes. The results displayed allow for inspection of members of the individual CUPP groups and include information about experimentally characterized members. The web server and the other resources on the CUPP platform can be accessed from https://cupp.info.


Assuntos
Metabolismo dos Carboidratos , Enzimas/química , Enzimas/genética , Anotação de Sequência Molecular , Software , Algoritmos , Enzimas/classificação , Enzimas/metabolismo , Internet , Peptídeos/química , Análise de Sequência de DNA , Análise de Sequência de Proteína
5.
Cancer Lett ; 473: 176-185, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-31923436

RESUMO

Altered cellular metabolism is a hallmark of cancer. Metabolic rewiring in cancer cells occurs due to the activation of oncogenes, inactivation of tumor suppressor genes, and/or other adaptive changes in cell signaling pathways. Furthermore, altered metabolism is also reported in tumor-corrupted stromal cells as a result of their interaction with cancer cells or due to their adaptation in the dynamic tumor microenvironment. Metabolic alterations are associated with dysregulation of metabolic enzymes and tumor-stromal metabolic crosstalk is vital for the progressive malignant journey of the tumor cells. Therefore, several therapies targeting metabolic enzymes have been evaluated and/or are being investigated in preclinical and clinical studies. In this review, we discuss some important metabolic enzymes that are altered in tumor and/or stromal cells, and focus on their role in supporting tumor growth. Moreover, we also discuss studies carried out in various cancers to target these metabolic abnormalities for therapeutic exploitation.


Assuntos
Carcinogênese/patologia , Inibidores Enzimáticos/farmacologia , Enzimas/metabolismo , Neoplasias/patologia , Células Estromais/enzimologia , Animais , Carcinogênese/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Enzimas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/patologia , Microambiente Tumoral/fisiologia
6.
Crit Rev Biotechnol ; 40(2): 231-246, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31914816

RESUMO

It is generally accepted that oligomeric enzymes evolve from their monomeric ancestors, and the evolution process generates superior structural benefits for functional advantages. Furthermore, adjusting the transition between different oligomeric states is an important mechanism for natural enzymes to regulate their catalytic functions for adapting environmental fluctuations in nature, which inspires researchers to mimic such a strategy to develop artificially oligomerized enzymes through protein engineering for improved performance under specific conditions. On the other hand, transforming oligomeric enzymes into their monomers is needed in fundamental research for deciphering catalytic mechanisms as well as exploring their catalytic capacities for better industrial applications. In this article, strategies for developing artificially oligomerized and monomerized enzymes are reviewed and highlighted by their applications. Furthermore, advances in the computational prediction of oligomeric structures are introduced, which would accelerate the systematic design of oligomeric and monomeric enzymes. Finally, the current challenges and future directions in this field are discussed.


Assuntos
Enzimas/genética , Engenharia de Proteínas , Bioengenharia , Catálise , Enzimas/química , Enzimas/metabolismo , Estabilidade Proteica
7.
PLoS Comput Biol ; 16(1): e1007600, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917825

RESUMO

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.


Assuntos
Enzimas , Método de Monte Carlo , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Especificidade por Substrato/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Azidas/química , Azidas/metabolismo , Sítios de Ligação/genética , Catálise , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Metionina tRNA Ligase/química , Metionina tRNA Ligase/genética , Metionina tRNA Ligase/metabolismo , Mutação/genética , Norleucina/análogos & derivados , Norleucina/química , Norleucina/metabolismo
8.
BMC Bioinformatics ; 21(1): 19, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937255

RESUMO

BACKGROUND: In order to improve the accuracy of constraint-based metabolic models, several approaches have been developed which intend to integrate additional biological information. Two of these methods, MOMENT and GECKO, incorporate enzymatic (kcat) parameters and enzyme mass constraints to further constrain the space of feasible metabolic flux distributions. While both methods have been proven to deliver useful extensions of metabolic models, they may considerably increase size and complexity of the models and there is currently no tool available to fully automate generation and calibration of such enzyme-constrained models from given stoichiometric models. RESULTS: In this work we present three major developments. We first conceived short MOMENT (sMOMENT), a simplified version of the MOMENT approach, which yields the same predictions as MOMENT but requires significantly fewer variables and enables direct inclusion of the relevant enzyme constraints in the standard representation of a constraint-based model. When measurements of enzyme concentrations are available, these can be included as well leading in the extreme case, where all enzyme concentrations are known, to a model representation that is analogous to the GECKO approach. Second, we developed the AutoPACMEN toolbox which allows an almost fully automated creation of sMOMENT-enhanced stoichiometric metabolic models. In particular, this includes the automatic read-out and processing of relevant enzymatic data from different databases and the reconfiguration of the stoichiometric model with embedded enzymatic constraints. Additionally, tools have been developed to adjust (kcat and enzyme pool) parameters of sMOMENT models based on given flux data. We finally applied the new sMOMENT approach and the AutoPACMEN toolbox to generate an enzyme-constrained version of the E. coli genome-scale model iJO1366 and analyze its key properties and differences with the standard model. In particular, we show that the enzyme constraints improve flux predictions (e.g., explaining overflow metabolism and other metabolic switches) and demonstrate, for the first time, that these constraints can markedly change the spectrum of metabolic engineering strategies for different target products. CONCLUSIONS: The methodological and tool developments presented herein pave the way for a simplified and routine construction and analysis of enzyme-constrained metabolic models.


Assuntos
Enzimas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Automação , Enzimas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Engenharia Metabólica , Redes e Vias Metabólicas , Modelos Biológicos
9.
Chem Soc Rev ; 49(1): 233-262, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31815263

RESUMO

Enzymes are versatile catalysts and their synthetic potential has been recognized for a long time. In order to exploit their full potential, enzymes often need to be re-engineered or optimized for a given application. (Semi-) rational design has emerged as a powerful means to engineer proteins, but requires detailed knowledge about structure function relationships. In turn, directed evolution methodologies, which consist of iterative rounds of diversity generation and screening, can improve an enzyme's properties with virtually no structural knowledge. Current diversity generation methods grant us access to a vast sequence space (libraries of >1012 enzyme variants) that may hide yet unexplored catalytic activities and selectivity. However, the time investment for conventional agar plate or microtiter plate-based screening assays represents a major bottleneck in directed evolution and limits the improvements that are obtainable in reasonable time. Ultrahigh-throughput screening (uHTS) methods dramatically increase the number of screening events per time, which is crucial to speed up biocatalyst design, and to widen our knowledge about sequence function relationships. In this review, we summarize recent advances in uHTS for directed enzyme evolution. We shed light on the importance of compartmentalization to preserve the essential link between genotype and phenotype and discuss how cells and biomimetic compartments can be applied to serve this function. Finally, we discuss how uHTS can inspire novel functional metagenomics approaches to identify natural biocatalysts for novel chemical transformations.


Assuntos
Evolução Molecular Direcionada , Enzimas/metabolismo , Ensaios de Triagem em Larga Escala , Biocatálise , Enzimas/química , Enzimas/genética , Engenharia de Proteínas
10.
Proc Natl Acad Sci U S A ; 117(1): 355-361, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31879352

RESUMO

The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designated Rceh1-3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that of ceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing the ceh1 mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the corresponding Rceh lines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.


Assuntos
Substituição de Aminoácidos , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Enzimas/genética , Oxirredutases/genética , Terpenos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Núcleo Celular/metabolismo , Enzimas/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Oxirredutases/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/genética , Plastídeos/metabolismo
11.
Nat Chem Biol ; 16(2): 197-205, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31844304

RESUMO

Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l-1 and a yield on glucose of 405.8 µg g-1. Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.


Assuntos
Endocanabinoides/biossíntese , Engenharia Metabólica/métodos , Ácidos Oleicos/biossíntese , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Coenzima A Ligases/genética , Endocanabinoides/genética , Enzimas/genética , Enzimas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Microrganismos Geneticamente Modificados , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Ácidos Oleicos/genética , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Fosfolipídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Food Chem ; 309: 125608, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31678673

RESUMO

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) can improve wound healing of potato tubers; however, how the chemical regulates reactive oxygen species (ROS) generation and scavenging during wound healing is not completely understood. BTH at 100 mg·L-1 regulated changes in ROS generation and scavenging in healing tissues of potato tubers. A higher H2O2 content was presented in healing tissues of potato tubers, while cell membrane permeability and malondialdehyde content declined due to BTH treatment. Additionally, the activities and transcript level of enzymes related with ROS generation, including NADPH oxidase, peroxidase and polyamine oxidase, as well as enzymes involved in ROS scavenging, such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, were significantly enhanced by BTH treatment. It is suggested that ROS metabolism might play a crucial role in wound healing of potato tubers mediated by BTH during postharvest.


Assuntos
Tubérculos/efeitos dos fármacos , Tubérculos/metabolismo , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/metabolismo , Tiadiazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
13.
PLoS Comput Biol ; 15(12): e1007569, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31869345

RESUMO

Rossmann folds are ancient, frequently diverged domains found in many biological reaction pathways where they have adapted for different functions. Consequently, discernment and classification of their homologous relations and function can be complicated. We define a minimal Rossmann-like structure motif (RLM) that corresponds for the common core of known Rossmann domains and use this motif to identify all RLM domains in the Protein Data Bank (PDB), thus finding they constitute about 20% of all known 3D structures. The Evolutionary Classification of protein structure Domains (ECOD) classifies RLM domains in a number of groups that lack evidence for homology (X-groups), which suggests that they could have evolved independently multiple times. Closely related, homologous RLM enzyme families can diverge to bind different ligands using similar binding sites and to catalyze different reactions. Conversely, non-homologous RLM domains can converge to catalyze the same reactions or to bind the same ligand with alternate binding modes. We discuss a special case of such convergent evolution that is relevant to the polypharmacology paradigm, wherein the same drug (methotrexate) binds to multiple non-homologous RLM drug targets with different topologies. Finally, assigning proteins with RLM domain to the Enzyme Commission classification suggest that RLM enzymes function mainly in metabolism (and comprise 38% of reference metabolic pathways) and are overrepresented in extant pathways that represent ancient biosynthetic routes such as nucleotide metabolism, energy metabolism, and metabolism of amino acids. In fact, RLM enzymes take part in five out of eight enzymatic reactions of the Wood-Ljungdahl metabolic pathway thought to be used by the last universal common ancestor (LUCA). The prevalence of RLM domains in this ancient metabolism might explain their wide distribution among enzymes.


Assuntos
Evolução Molecular , Domínios Proteicos/genética , Sítios de Ligação/genética , Domínio Catalítico/genética , Biologia Computacional , Bases de Dados de Proteínas , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Humanos , Ligantes , Redes e Vias Metabólicas/genética , Modelos Moleculares , Ligação Proteica/genética , Software , Homologia Estrutural de Proteína
14.
Parasit Vectors ; 12(1): 584, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842984

RESUMO

BACKGROUND: Blood meal host selection by mosquito vectors is an important component in understanding disease dynamics of pathogens that threaten endemic fauna in isolated islands such as Galápagos. Research on the feeding behavior of mosquitoes can provide clues to the hosts and vectors involved in disease transmission. This information is particularly critical for endemic wildlife fauna in island systems that have evolved without resistance to novel diseases such as avian malaria. The aims of this study were to determine the blood-feeding patterns of two species of mosquitoes found in Galápagos and discuss how their feeding behavior may influence the transmission of pathogens such as avian malaria. METHODS: In the summer of 2015, we sampled two mosquito species (Aedes taeniorhynchus and Culex quinquefasciatus) across 18 different sites on Isla Santa Cruz, which is the second largest island in Galápagos and has the largest human population. We trapped mosquitoes using CDC light traps and CDC gravid traps and identified sources of blood meals for engorged mosquitoes by sequencing a portion of the vertebrate mitochondrial cytochrome b gene. RESULTS: Out of 947 female mosquitoes captured, 320 were blood-fed, and PCR amplifications were successful for 301 of the blood meals. Results revealed that both Aedes taeniorhynchus and Culex quinquefasciatus feed from a variety of vertebrate taxa, numerically dominated by humans on Isla Santa Cruz. CONCLUSIONS: The high proportion of mammalian blood meals could represent locally available and abundant hosts on Santa Cruz. However, host surveys and estimates of relative abundances of vertebrate species will need to accompany mosquito trapping studies on non-inhabited and inhabited islands in Galápagos to further validate this.


Assuntos
Aedes/fisiologia , Culex/fisiologia , Comportamento Alimentar , Mosquitos Vetores/fisiologia , Animais , Citocromos b/genética , Equador , Entomologia/métodos , Enzimas/sangue , Enzimas/genética , Técnicas de Genotipagem/métodos , Humanos , Mamíferos
15.
Nat Chem Biol ; 15(12): 1148-1155, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740832

RESUMO

Archaeosine (G+), 7-formamidino-7-deazaguanosine, is an archaea-specific modified nucleoside found at the 15th position of tRNAs. In Euryarchaeota, 7-cyano-7-deazaguanine (preQ0)-containing tRNA (q0N-tRNA), synthesized by archaeal tRNA-guanine transglycosylase (ArcTGT), has been believed to be converted to G+-containing tRNA (G+-tRNA) by the paralog of ArcTGT, ArcS. However, we found that several euryarchaeal ArcSs have lysine transfer activity to q0N-tRNA to form q0kN-tRNA, which has a preQ0 lysine adduct as a base. Through comparative genomics and biochemical experiments, we found that ArcS forms a robust complex with a radical S-adenosylmethionine (SAM) enzyme named RaSEA. The ArcS-RaSEA complex anaerobically converted q0N-tRNA to G+-tRNA in the presence of SAM and lysine via q0kN-tRNA. We propose that ArcS and RaSEA should be considered an archaeosine synthase α-subunit (lysine transferase) and ß-subunit (q0kN-tRNA lyase), respectively.


Assuntos
Enzimas/metabolismo , Guanosina/análogos & derivados , S-Adenosilmetionina/metabolismo , Bases de Dados Genéticas , Enzimas/genética , Perfilação da Expressão Gênica , Guanosina/biossíntese , Lisina/metabolismo , Especificidade por Substrato
16.
Sheng Wu Gong Cheng Xue Bao ; 35(10): 1806-1818, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31668030

RESUMO

Industrial enzymes are the "chip" of modern bio-industries, supporting tens- and hundreds-fold of downstream industries development. Elucidating the relationships between enzyme structures and functions is fundamental for industrial applications. Recently, with the advanced developments of protein crystallization and computational simulation technologies, the structure-function relationships have been extensively studied, making the rational design and de novo design become possible. This paper reviews the progress of structure-function relationships of industrial enzymes and applications, and address future developments.


Assuntos
Biotecnologia , Enzimas/química , Enzimas/metabolismo , Engenharia Metabólica , Engenharia de Proteínas , Biocatálise , Enzimas/genética , Relação Estrutura-Atividade
17.
Sheng Wu Gong Cheng Xue Bao ; 35(10): 1819-1828, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31668031

RESUMO

We review major computational chemistry techniques applied in industrial enzyme studies, especially approaches intended for guiding enzyme engineering. These include molecular mechanics force field and molecular dynamics simulation, quantum mechanical and combined quantum mechanical/molecular mechanical approaches, electrostatic continuum models, molecular docking, etc. These approaches are essentially introduced from the following two angles for viewing: one is about the methods themselves, including the basic concepts, the primary computational results, and potential advantages and limitations; the other is about obtaining valuable information from the respective calculations to guide the design of mutants and mutant libraries.


Assuntos
Enzimas/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Teoria Quântica , Enzimas/química , Enzimas/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Eletricidade Estática
18.
Proc Natl Acad Sci U S A ; 116(43): 21493-21500, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31591248

RESUMO

Chemoproteomic methods can report directly on endogenous, active enzyme populations, which can differ greatly from measures of transcripts or protein abundance alone. Detection and quantification of family-wide probe engagement generally requires LC-MS/MS or gel-based detection methods, which suffer from low resolution, significant input proteome requirements, laborious sample preparation, and expensive equipment. Therefore, methods that can capitalize on the broad target profiling capacity of family-wide chemical probes but that enable specific, rapid, and ultrasensitive quantitation of protein activity in native samples would be useful for basic, translational, and clinical proteomic applications. Here we develop and apply a method that we call soluble activity-dependent proximity ligation (sADPL), which harnesses family-wide chemical probes to convert active enzyme levels into amplifiable barcoded oligonucleotide signals. We demonstrate that sADPL coupled to quantitative PCR signal detection enables multiplexed "writing" and "reading" of active enzyme levels across multiple protein families directly at picogram levels of whole, unfractionated proteome. sADPL profiling in a competitive format allows for highly sensitive detection of drug-protein interaction profiling, which allows for direct quantitative measurements of in vitro and in vivo on- and off-target drug engagement. Finally, we demonstrate that comparative sADPL profiling can be applied for high-throughput molecular phenotyping of primary human tumor samples, leading to the discovery of new connections between metabolic and proteolytic enzyme activity in specific tumor compartments and patient outcomes. We expect that this modular and multiplexed chemoproteomic platform will be a general approach for drug target engagement, as well as comparative enzyme activity profiling for basic and clinical applications.


Assuntos
Cromatografia Líquida/métodos , Enzimas/química , Proteoma/química , Proteômica/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular Tumoral , Enzimas/genética , Enzimas/metabolismo , Humanos , Neoplasias/química , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/metabolismo , Proteoma/genética , Proteoma/metabolismo , Sensibilidade e Especificidade
19.
J Agric Food Chem ; 67(38): 10624-10636, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31483633

RESUMO

The freshness and color quality of postharvest tea leaves can be markedly prolonged and retained by proper preservation measures. Here, we investigated the dynamic changes of chlorophyll and its derivatives in postharvest tea leaves under different low-temperature treatments using natural withering as a control. Chlorophyll decomposition was found closely related with chlorophyllide, pheophorbide, and pheophytin. Low-temperature withering could slow chlorophyll degradation in postharvest tea leaves via significant inhibition on the enzyme activity and gene expression of Mg-dechelatase, chlorophyllase, and pheophorbide a oxygenase. At the initial stage of withering, a significant increase was observed in the chlorophyll content, expression of chlorophyll-synthesis-related enzymes (such as glutamyl-tRNA synthetase, etc.), and chlorophyll synthase activity in newly picked tea leaves. Moreover, an obvious decrease was found in the content of l-glutamate as the foremost precursor substance of chlorophyll synthesis. Hence, our findings revealed that the chlorophyll synthesis reaction was induced by the light-dehydration-stress in the initial withering of tea leaves. This study provides a theoretical basis for exploring preservation technology in actual green tea production.


Assuntos
Camellia sinensis/genética , Camellia sinensis/metabolismo , Clorofila/metabolismo , Manipulação de Alimentos/métodos , Regulação da Expressão Gênica de Plantas , Camellia sinensis/química , Camellia sinensis/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Clorofila/química , Cor , Enzimas/genética , Enzimas/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Temperatura
20.
Elife ; 82019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31532392

RESUMO

Functional conservation is known to constrain protein evolution. Nevertheless, the long-term divergence patterns of proteins maintaining the same molecular function and the possible limits of this divergence have not been explored in detail. We investigate these fundamental questions by characterizing the divergence between ancient protein orthologs with conserved molecular function. Our results demonstrate that the decline of sequence and structural similarities between such orthologs significantly slows down after ~1-2 billion years of independent evolution. As a result, the sequence and structural similarities between ancient orthologs have not substantially decreased for the past billion years. The effective divergence limit (>25% sequence identity) is not primarily due to protein sites universally conserved in all linages. Instead, less than four amino acid types are accepted, on average, per site across orthologous protein sequences. Our analysis also reveals different divergence patterns for protein sites with experimentally determined small and large fitness effects of mutations. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).


Assuntos
Enzimas/genética , Enzimas/metabolismo , Evolução Molecular , Redes e Vias Metabólicas/genética , Biologia Computacional , Enzimas/química , Conformação Proteica
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