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1.
Molecules ; 26(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34443596

RESUMO

FAH domain containing protein 1 (FAHD1) acts as oxaloacetate decarboxylase in mitochondria, contributing to the regulation of the tricarboxylic acid cycle. Guided by a high-resolution X-ray structure of FAHD1 liganded by oxalate, the enzymatic mechanism of substrate processing is analyzed in detail. Taking the chemical features of the FAHD1 substrate oxaloacetate into account, the potential inhibitor structures are deduced. The synthesis of drug-like scaffolds afforded first-generation FAHD1-inhibitors with activities in the low micromolar IC50 range. The investigations disclosed structures competing with the substrate for binding to the metal cofactor, as well as scaffolds, which may have a novel binding mode to FAHD1.


Assuntos
Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Humanos , Hidrolases/química , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica
2.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
3.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299249

RESUMO

Melanoma as a very aggressive type of cancer is still in urgent need of improved treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and arginine deiminase (ADI-PEG20) are two of many suggested drugs for treating melanoma. Both have shown anti-tumor activities without harming normal cells. However, resistance to both drugs has also been noted. Studies on the mechanism of action of and resistance to these drugs provide multiple targets that can be utilized to increase the efficacy and overcome the resistance. As a result, combination strategies have been proposed for these drug candidates with various other agents, and achieved enhanced or synergistic anti-tumor effect. The combination of TRAIL and ADI-PEG20 as one example can greatly enhance the cytotoxicity to melanoma cells including those resistant to the single component of this combination. It is found that combination treatment generally can alter the expression of the components of cell signaling in melanoma cells to favor cell death. In this paper, the signaling of TRAIL and ADI-PEG20-induced arginine deprivation including the main mechanism of resistance to these drugs and exemplary combination strategies is discussed. Finally, factors hampering the clinical application of both drugs, current and future development to overcome these hurdles are briefly discussed.


Assuntos
Hidrolases/farmacologia , Melanoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Arginina/deficiência , Arginina/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Hidrolases/metabolismo , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
4.
Fish Physiol Biochem ; 47(4): 1211-1227, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34173183

RESUMO

Cichlasoma dimerus is a neotropical cichlid that has been used as a biological model for neuroendocrinology studies. However, its culture is problematic in terms of larval feeding to allow having enough fry quantity and quality. Larviculture requires full knowledge about the digestive system and nutrition; therefore, this study was intended to assess the digestive enzymes' changes at different ages during the early ontogeny. Acid protease activity was detectable from the first day after hatching (dah), increasing to its maximum peaks on 9 dah. In contrast, alkaline proteases had low activity in the first days of life but reached their maximum activity on 17 dah. Chymotrypsin, L-aminopeptidase, and carboxypeptidase A activities increased at 6 dah, while trypsin activity was first detected on 13 dah and reached its maximum activity on 17 dah. Lipase and α-amylase activity were detectable at low levels in the first days of life, but the activity fluctuated and reaching its maximum activity at 21 dah. Alkaline phosphatase continued to oscillate and had two maximum activity peaks, the first at 6 dah and the second at 19 dah. Zymograms of alkaline proteases on day 6 dah six revealed four activity bands with molecular weights from 16.1 to 77.7 kDa. On 13 dah, two more activity bands of 24.4 and 121.9 kDa were detected, having a total of six proteases. The enzymatic activity analyzes indicate the digestive system shows the low activity of some enzymes in the first days after hatching, registering significant increases on 6 dah and the maximum peaks of activities around at 17 dah. Therefore, we recommend replacing live food with dry feed and only providing dry feed after day 17 dah.


Assuntos
Ciclídeos/crescimento & desenvolvimento , Ciclídeos/metabolismo , Hidrolases/metabolismo , Animais , Digestão , Larva/crescimento & desenvolvimento , Larva/metabolismo
5.
Nat Commun ; 12(1): 4021, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188040

RESUMO

Chlamydiae are highly successful strictly intracellular bacteria associated with diverse eukaryotic hosts. Here we analyzed metagenome-assembled genomes of the "Genomes from Earth's Microbiomes" initiative from diverse environmental samples, which almost double the known phylogenetic diversity of the phylum and facilitate a highly resolved view at the chlamydial pangenome. Chlamydiae are defined by a relatively large core genome indicative of an intracellular lifestyle, and a highly dynamic accessory genome of environmental lineages. We observe chlamydial lineages that encode enzymes of the reductive tricarboxylic acid cycle and for light-driven ATP synthesis. We show a widespread potential for anaerobic energy generation through pyruvate fermentation or the arginine deiminase pathway, and we add lineages capable of molecular hydrogen production. Genome-informed analysis of environmental distribution revealed lineage-specific niches and a high abundance of chlamydiae in some habitats. Together, our data provide an extended perspective of the variability of chlamydial biology and the ecology of this phylum of intracellular microbes.


Assuntos
Chlamydia/genética , Ciclo do Ácido Cítrico/genética , Genoma Bacteriano/genética , Metagenoma/genética , Acanthamoeba/microbiologia , Animais , Chlamydia/classificação , Chlamydia/isolamento & purificação , Ecossistema , Peixes/parasitologia , Brânquias/parasitologia , Hidrolases/metabolismo , Filogenia , Ácido Pirúvico/metabolismo , Sequenciamento Completo do Genoma
6.
Environ Sci Technol ; 55(14): 9817-9825, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34080849

RESUMO

Fluorochemicals are persistent, bioaccumulative, and toxic compounds that are widely tributed in the environment. Developing efficient biodegradation strategies to decompose the fluorochemicals via breaking the inert C-F bonds presents a holistic challenge. As a promising biodegradation enzyme candidate, fluoroacetate dehalogenase (FAcD) has been reported as the only non-metallic enzyme to catalyze the cleavage of the strong C-F bond. Here, we systematically investigated the catalytic actions of FAcD toward its natural substrate fluoroacetate using molecular dynamics simulations and quantum mechanism/molecular mechanism calculations. We propose that the enzymatic transformation involves four elementary steps, (I) C-F bond activation, (II) nucleophilic attack, (III) C-O bond cleavage, and (IV) proton transfer. Our results show that nucleophilic attack is the rate-determining step. However, for difluoroacetate and trifluoroacetate, C-F bond activation, instead of nucleophilic attack, becomes the rate-determining step. We show that FAcD, originally recognized as α-fluorocarboxylic acid degradation enzyme, can catalyze the defluorination of difluoroacetate to glyoxylate, which is captured by our high-resolution mass spectrometry experiments. In addition, we employed amino acid electrostatic analysis method to screen potential mutation hotspots for tuning FAcD's electrostatic environment to favor substrate conversion. The comprehensive understanding of catalytic mechanism will inform a rational enzyme engineering strategy to degrade fluorochemicals for benefits of environmental sustainability.


Assuntos
Hidrolases , Simulação de Dinâmica Molecular , Aminoácidos , Catálise , Hidrolases/metabolismo , Teoria Quântica
7.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072602

RESUMO

Enzymes play a fundamental role in many biological processes. We present a theoretical approach to investigate the catalytic power of the haloalkane dehalogenase reaction with 1,2-dichloroethane. By removing the three main active-site residues one by one from haloalkane dehalogenase, we found two reactive descriptors: one descriptor is the distance difference between the breaking bond and the forming bond, and the other is the charge difference between the transition state and the reactant complex. Both descriptors scale linearly with the reactive barriers, with the three-residue case having the smallest barrier and the zero-residue case having the largest. The results demonstrate that, as the number of residues increases, the catalytic power increases. The predicted free energy barriers using the two descriptors of this reaction in water are 23.1 and 24.2 kcal/mol, both larger than the ones with any residues, indicating that the water solvent hinders the reactivity. Both predicted barrier heights agree well with the calculated one at 25.2 kcal/mol using a quantum mechanics and molecular dynamics approach, and also agree well with the experimental result at 26.0 kcal/mol. This study shows that reactive descriptors can also be used to describe and predict the catalytic performance for enzyme catalysis.


Assuntos
Dicloretos de Etileno/química , Dicloretos de Etileno/farmacologia , Hidrolases/química , Hidrolases/metabolismo , Algoritmos , Catálise , Ativação Enzimática/efeitos dos fármacos , Modelos Químicos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Teoria Quântica , Relação Estrutura-Atividade
8.
Sci Rep ; 11(1): 12410, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127732

RESUMO

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and H2O2, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer. For extended self-disinfection, however, active and stable AcT bound onto or incorporated into a surface coating is necessary. In the current study, an active, stable and reusable AcT-based coating was developed by incorporating AcT into a polydopamine (PDA) matrix in a single step, thereby forming a biocatalytic composite onto a variety of surfaces. The resulting AcT-PDA composite coatings on glass, metal and epoxy surfaces yielded up to 7-log reduction of Gram-positive and Gram-negative bacteria when in contact with the biocatalytic coating. This composite coating also possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing. As a result, this enzyme-polymer composite technique may serve as a general strategy for preparing antibacterial and antiviral surfaces for applications in health care and common infrastructure safety, such as in schools, the workplace, transportation, etc.


Assuntos
Antibacterianos/química , Antivirais/química , Proteínas de Bactérias/química , Hidrolases/química , Indóis/química , Polímeros/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , COVID-19/patologia , COVID-19/virologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/metabolismo , Materiais Revestidos Biocompatíveis/farmacologia , Estabilidade de Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Cinética , Mycobacterium smegmatis/enzimologia , Ácido Peracético/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/efeitos dos fármacos
9.
Nat Commun ; 12(1): 3867, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162839

RESUMO

Enzymes can evolve new catalytic activity when environmental changes present them with novel substrates. Despite this seemingly straightforward relationship, factors other than the direct catalytic target can also impact adaptation. Here, we characterize the catalytic activity of a recently evolved bacterial methyl-parathion hydrolase for all possible combinations of the five functionally relevant mutations under eight different laboratory conditions (in which an alternative divalent metal is supplemented). The resultant adaptive landscapes across this historical evolutionary transition vary in terms of both the number of "fitness peaks" as well as the genotype(s) at which they are found as a result of genotype-by-environment interactions and environment-dependent epistasis. This suggests that adaptive landscapes may be fluid and molecular adaptation is highly contingent not only on obvious factors (such as catalytic targets), but also on less obvious secondary environmental factors that can direct it towards distinct outcomes.


Assuntos
Adaptação Fisiológica/genética , Bactérias/genética , Proteínas de Bactérias/genética , Epistasia Genética , Hidrolases/genética , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Evolução Molecular , Interação Gene-Ambiente , Genótipo , Hidrolases/química , Hidrolases/metabolismo , Cinética , Metais/química , Metais/metabolismo , Metil Paration/química , Metil Paration/metabolismo , Mutação , Domínios Proteicos , Homologia de Sequência de Aminoácidos
10.
Arch Biochem Biophys ; 705: 108896, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33940035

RESUMO

The evolution of enzyme catalytic structures and mechanisms has drawn increasing attention. In this study, we investigate the functional divergence from phosphomonoesterase to inorganic pyrophosphatase in the haloacid dehalogenase (HAD) superfamily. In this study, a series of models was constructed, and calculations were performed by using density functional theory with the B3LYP functional. The calculations suggest that in most HAD members, the active-site structure is unstable due to the binding of the substrate inorganic pyrophosphate (PPi), and reactions involving PPi cannot be catalyzed. In BT2127, which is a unique member of the HAD superfamily, the Mg2+-coordinating residues Asn172 and Glu47 play a role in stabilizing the active-site structure to adapt to the substrate PPi by providing much stronger coordination interactions with the Mg2+ ion. The calculation results suggest that Asn172 and Glu47 are crucial in the evolution of the inorganic pyrophosphatase activity in the HAD superfamily. Our study provides definitive chemical insight into the functional divergence of the HAD superfamily, and helps in understanding the evolution of enzyme catalytic structures and mechanisms.


Assuntos
Hidrolases/metabolismo , Pirofosfatase Inorgânica/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Biocatálise , Domínio Catalítico , Hidrolases/química , Cinética , Magnésio/metabolismo , Modelos Moleculares , Especificidade por Substrato
11.
Microbes Environ ; 36(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34024869

RESUMO

Carbonyl sulfide (COS) is the most abundant and long-lived sulfur-containing gas in the atmosphere. Soil is the main sink of COS in the atmosphere and uptake is dominated by soil microorganisms; however, biochemical research has not yet been conducted on fungal COS degradation. COS hydrolase (COSase) was purified from Trichoderma harzianum strain THIF08, which degrades COS at concentrations higher than 10,000 parts per million by volume from atmospheric concentrations, and its gene cos (492 bp) was cloned. The recombinant protein purified from Escherichia coli expressing the cos gene converted COS to H2S. The deduced amino acid sequence of COSase (163 amino acids) was assigned to clade D in the phylogenetic tree of the ß-carbonic anhydrase (ß-CA) family, to which prokaryotic COSase and its structurally related enzymes belong. However, the COSase of strain THIF08 differed from the previously known prokaryotic COSase and its related enzymes due to its low reactivity to CO2 and inability to hydrolyze CS2. Sequence comparisons of the active site amino acids of clade D ß-CA family enzymes suggested that various Ascomycota, particularly Sordariomycetes and Eurotiomycetes, possess similar enzymes to the COSase of strain THIF08 with >80% identity. These fungal COSase were phylogenetically distant to prokaryotic clade D ß-CA family enzymes. These results suggest that various ascomycetes containing COSase contribute to the uptake of COS by soil.


Assuntos
Anidrases Carbônicas/química , Proteínas Fúngicas/química , Hidrolases/química , Hypocreales/enzimologia , Óxidos de Enxofre/metabolismo , Sequência de Aminoácidos , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Hypocreales/química , Hypocreales/genética , Filogenia , Alinhamento de Sequência , Solo/química
12.
Planta ; 253(6): 122, 2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34003383

RESUMO

MAIN CONCLUSION: The rice OsFAH gene functions identically to that of Arabidopsis SSCD1 encoding FAH. Loss of OsFAH causes rice sterility. Fumarylacetoacetate hydrolase (FAH) is the last enzyme in the tyrosine (Tyr) degradation pathway that is crucial for animals. By genetic analysis of the mutant of Short-day Sensitive Cell Death 1 gene encoding Arabidopsis FAH, we first found the pathway also plays a critical role in plants (Han et al., Plant Physiol 162:1956-1964, 2013). To further understand the role of the Tyr degradation pathway in plants, we investigated a biological function of the rice FAH. Firstly, the cDNA of rice FAH gene (OsFAH) was cloned and confirmed to be able to rescue the Arabidopsis Short-day Sensitive Cell Death 1 mutant defective in the FAH. Then, we identified the OsFAH T-DNA insertion mutant and generated the OsFAH RNA interference lines, and found that loss of OsFAH results in rice sterility. Furthermore, we analyzed expression of the OsFAH gene in roots, stems, leaves and young panicles at booting stage of rice and found that its transcript level was highest in young panicles and lowest in roots. In addition, the expression analysis of ß-glucuronidase driven by OsFAH promoter in transgenic Arabidopsis showed that the OsFAH promoter was highly active in aerial tissues in vegetative stage, and sepals, filaments and stigma in reproductive stage. These results suggested that FAH plays an important role in rice fertility.


Assuntos
Arabidopsis , Oryza , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Fertilidade , Regulação da Expressão Gênica de Plantas , Hidrolases/genética , Hidrolases/metabolismo , Oryza/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas/metabolismo
13.
Sheng Wu Gong Cheng Xue Bao ; 37(4): 1298-1311, 2021 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-33973443

RESUMO

As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral ß-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0-8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.


Assuntos
Rhodospirillaceae , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Especificidade por Substrato
14.
Gene ; 792: 145736, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34051337

RESUMO

BACKGROUND: The aim of this study was to explore the expression levels and biological significance of TDO2 in colorectal cancer (CRC). METHODS: First, we explored the potential oncogenic roles of TDO2 across 33 tumors based on data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Second, we evaluated TDO2 protein expression in 55 CRC tissue samples and 30 cDNA samples by immunohistochemistry and qPCR. Third, we investigated the effect of TDO2 on CRC cells by cell proliferation, wound healing, invasion, and colony formation assays. Finally, we determined the protein that is most closely associated with TDO2 via bioinformatics analysis, enriched the key pathways, and verified them. RESULTS: The expression level of TDO2 was found to be associated with the tumor clinical stage in CRC. A high expression of TDO2 was associated with a poor outcome in CRC patients. Inhibition of TDO2 expression by RNAi in LoVo and HCT116 cell lines significantly reduced the proliferation, migration, and invasion abilities as well as colony formation abilities of cells. Further, knockdown of TDO2 expression induced inactivation of the TDO2-KYNU-AhR signaling pathway. CONCLUSION: The results suggest that TDO2 plays an important role in the progression of CRC. Accordingly, TDO2 is a potential therapeutic target in CRC.


Assuntos
Adenocarcinoma/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinogênese/genética , Neoplasias Colorretais/genética , Hidrolases/genética , Receptores de Hidrocarboneto Arílico/genética , Triptofano Oxigenase/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Atlas como Assunto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Hidrolases/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Curva ROC , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Análise de Sobrevida , Triptofano Oxigenase/antagonistas & inibidores , Triptofano Oxigenase/metabolismo
15.
Plant Physiol ; 185(4): 1559-1573, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33793956

RESUMO

The presence of mixed-linkage (1,3;1,4)-ß-d-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.


Assuntos
Parede Celular/metabolismo , Escuridão , Glucanos/metabolismo , Hidrolases/metabolismo , Folhas de Planta/metabolismo , Polissacarídeos/metabolismo , Zea mays/genética , Zea mays/metabolismo , Parede Celular/genética , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Glucanos/genética , Hidrolases/genética , Mutação , Folhas de Planta/genética , Polissacarídeos/genética
16.
Bioconjug Chem ; 32(5): 964-970, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-33856762

RESUMO

HaloTag is a modified haloalkane dehalogenase used for many applications in chemical biology including protein purification, cell-based imaging, and cytosolic penetration assays. While working with purified, recombinant HaloTag protein, we discovered that HaloTag forms an internal disulfide bond under oxidizing conditions. In this work, we describe this internal disulfide formation and the conditions under which it occurs, and we identify the relevant cysteine residues. Further, we develop a mutant version of HaloTag, HaloTag8, that maintains activity while avoiding internal disulfide formation altogether. While there is no evidence that HaloTag is prone to disulfide formation in intracellular environments, researchers using recombinant HaloTag, HaloTag expressed on the cell surface, or HaloTag in the extracellular space might consider using HaloTag8 to avoid intramolecular disulfide formation.


Assuntos
Dissulfetos/química , Linhagem Celular , Cisteína/química , Hidrolases/química , Hidrolases/metabolismo
17.
Carbohydr Polym ; 262: 117943, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838820

RESUMO

Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus.


Assuntos
Aspergillus/enzimologia , Filtração/métodos , Sucos de Frutas e Vegetais , Proteínas Fúngicas/metabolismo , Ramnogalacturonanos/metabolismo , Metabolismo dos Carboidratos , Celulases/metabolismo , Manipulação de Alimentos/métodos , Glicosídeo Hidrolases/metabolismo , Hidrolases/metabolismo , Malus , Pectinas/metabolismo , Poligalacturonase/metabolismo , Proteômica
18.
Nat Chem Biol ; 17(8): 856-864, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33927411

RESUMO

Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.


Assuntos
Membrana Celular/metabolismo , Hidrolases/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas ras/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Leucemia Mieloide Aguda/patologia , Leucemia Promielocítica Aguda/patologia , Lipoilação , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular
19.
Biochemistry ; 60(16): 1312-1324, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33827210

RESUMO

The serine hydrolase (SH) superfamily is, perhaps, one of the largest functional enzyme classes in all forms of life and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of chemical proteomics techniques, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlases for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, we initially report a bioinformatics analysis toward the identification and categorization of nonredundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the full complement of SH activities during various developmental stages and in different adult tissues of Drosophila. Finally, as a proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult D. melanogaster, and we propose new research directions, resources, and tools that this study can provide to the fly community.


Assuntos
Drosophila melanogaster/enzimologia , Hidrolases/metabolismo , Serina/metabolismo , Animais , Domínio Catalítico , Hidrolases/química , Hidrólise , Modelos Moleculares , Proteômica
20.
Chem Asian J ; 16(11): 1382-1387, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33886165

RESUMO

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A-D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.


Assuntos
Hidrolases/metabolismo , Peptídeos Cíclicos/biossíntese , Aminoácidos/metabolismo , Sítios de Ligação , Citrulina/metabolismo , Hidrolases/classificação , Hidrolases/genética , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ornitina/metabolismo , Filogenia , Especificidade por Substrato
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