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1.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614218

RESUMO

The xyloglucan endotransglucosylase/hydrolase (XET/XEH, also named XTH) family is a multigene family, the function of which plays a significant role in cell-wall rebuilding and stress tolerance in plants. However, the specific traits of the XTH gene family members and their expression pattern in different tissues and under stress have not been carried out in sweet potato. Thirty-six XTH genes were identified in I. batatas, all of which had conserved structures (Glyco_hydro_16). Based on Neighbor-Joining phylogenetic analysis the IbXTHs can be divided into three subfamilies-the I/II, IIIA, and IIIB subfamilies, which were unevenly distributed on 13 chromosomes, with the exception of Chr9 and Chr15. Multiple cis-acting regions related to growth and development, as well as stress responses, may be found in the IbXTH gene promoters. The segmental duplication occurrences greatly aided the evolution of IbXTHs. The results of a collinearity analysis showed that the XTH genes of sweet potato shared evolutionary history with three additional species, including A. thaliana, G. max, and O. sativa. Additionally, based on the transcriptome sequencing data, the results revealed that the IbXTHs have different expression patterns in leaves, stems, the root body (RB), the distal end (DE), the root stock (RS), the proximal end (PE), the initiative storage root (ISR), and the fibrous root (FR), and many of them are well expressed in the roots. Differentially expressed gene (DEG) analysis of FRs after hormone treatment of the roots indicated that IbXTH28 and IbXTH30 are up-regulated under salicylic acid (SA) treatment but down-regulated under methyl jasmonate (MeJA) treatment. Attentionally, there were only two genes showing down-regulation under the cold and drought treatment. Collectively, all of the findings suggested that genes from the XTH family are crucial for root specificity. This study could provide a theoretical basis for further research on the molecular function of sweet potato XTH genes.


Assuntos
Ipomoea batatas , Ipomoea batatas/genética , Ipomoea batatas/metabolismo , Filogenia , Glicosiltransferases/metabolismo , Hidrolases/metabolismo , Regulação da Expressão Gênica de Plantas
2.
Commun Biol ; 6(1): 39, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36639437

RESUMO

The large-scale preparation of Polyehylene terephthalate (PET) hydrolysing enzymes in low-cost is critical for the biodegradation of PET in industry. In the present study, we demonstrate that the post-translational glycosylation of Pichia pastoris makes it a remarkable host for the heterologous expression of PETase from Ideonella sakaiensis 201-F6 (IsPETase). Taking advantage of the abundant N- and O-linked glycosylation sites in IsPETase and the efficient post-translational modification in endoplasmic reticulum, IsPETase is heavily glycosylated during secretory expression with P. pastoris, which improves the specific activity and thermostability of the enzyme dramatically. Moreover, the specific activity of IsPETase increased further after the bulky N-linked polysaccharide chains were eliminated by Endo-ß-N-acetylglucosaminidase H (Endo H). Importantly, the partially deglycosylated IsPETase still maintained high thermostability because of the remaining mono- and oligo-saccharide residues on the protein molecules. Consequently, the partially deglycosylated IsPETase was able to be applied at 50 °C and depolymerized raw, untreated PET flakes completely in 2 to 3 days. This platform was also applied for the preparation of a famous variant of IsPETase, Fast-PETase, and the same result was achieved. Partially deglycosylated Fast-PETase demonstrates elevated efficiency in degrading postconsumer-PET trays under 55 °C than 50 °C, the reported optimal temperature of Fast-PETase. The present study provides a strategy to modulate thermostable IsPETase through glycosylation engineering and paves the way for promoting PET biodegradation from laboratories to factories.


Assuntos
Burkholderiales , Hidrolases , Hidrolases/química , Burkholderiales/metabolismo , Processamento de Proteína Pós-Traducional , Polissacarídeos
3.
Sci Rep ; 13(1): 1267, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36690710

RESUMO

Agitation is a commonly encountered stress for enzymes during all stages of production and application, but investigations that aim to improve their tolerance using topological engineering have yet to be reported. Here, the plastic-degrading enzyme IsPETase was cyclized in a range of topologies including a cyclic monomer, cyclic dimer and catenane using SpyTag/SpyCatcher technologies, and their tolerance towards different stresses including mechanical agitation was investigated. The cyclic dimer and catenane topologies were less susceptible to agitation-induced inactivation resulting in enhancement of polyethylene terephthalate (PET) degradation. While contrary to conventional belief, cyclic topologies did not improve tolerance of IsPETase towards heat or proteolytic treatment, the close proximity of active sites in the dimeric and catenane variants was found to enhance PET conversion into small soluble products. Together, these findings illustrate that it is worthwhile to explore the topology engineering of enzymes used in heterogeneous catalysis as it improves factors that are often overlooked in homogeneous catalysis studies.


Assuntos
Catenanos , Polietilenotereftalatos , Polietilenotereftalatos/química , Plásticos , Hidrolases/metabolismo , Temperatura Alta
4.
BMC Plant Biol ; 23(1): 2, 2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36588160

RESUMO

BACKGROUND: Methane (CH4) and brassinosteroids (BRs) are important signaling molecules involved in a variety of biological processes in plants. RESULTS: Here, marigold (Tagetes erecta L. 'Marvel') was used to investigate the role and relationship between CH4 and BRs during adventitious root (AR) formation. The results showed a dose-dependent effect of CH4 and BRs on rooting, with the greatest biological effects of methane-rich water (MRW, CH4 donor) and 2,4-epibrassinolide (EBL) at 20% and 1 µmol L- 1, respectively. The positive effect of MRW on AR formation was blocked by brassinoazole (Brz, a synthetic inhibitor of EBL), indicating that BRs might be involved in MRW-regulated AR formation. MRW promoted EBL accumulation during rooting by up-regulating the content of campestanol (CN), cathasterone (CT), and castasterone (CS) and the activity of Steroid 5α-reductase (DET2), 22α-hydroxylase (DWF4), and BR-6-oxidase (BR6ox), indicating that CH4 could induce endogenous brassinolide (BR) production during rooting. Further results showed that MRW and EBL significantly down-regulated the content of cellulose, hemicellulose and lignin during rooting and significantly up-regulated the hydrolase activity, i.e. cmcase, xylanase and laccase. In addition, MRW and EBL also significantly promoted the activity of two major cell wall relaxing factors, xyloglucan endotransglucosylase/hydrolase (XTH) and peroxidase, which in turn promoted AR formation. While, Brz inhibited the role of MRW on these substances. CONCLUSIONS: BR might be involved in CH4-promoted AR formation by increasing cell wall relaxation.


Assuntos
Brassinosteroides , Celulose , Brassinosteroides/farmacologia , Metano/farmacologia , Hidrolases , Raízes de Plantas/fisiologia
5.
Cells ; 12(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36611984

RESUMO

GBA gene variants were the first genetic risk factor for Parkinson's disease. GBA encodes the lysosomal enzyme glucocerebrosidase (GBA), which is involved in sphingolipid metabolism. GBA exhibits a complex physiological function that includes not only the degradation of its substrate glucosylceramide but also the metabolism of other sphingolipids and additional lipids such as cholesterol, particularly when glucocerebrosidase activity is deficient. In the context of Parkinson's disease associated with GBA, the loss of GBA activity has been associated with the accumulation of α-synuclein species. In recent years, several hypotheses have proposed alternative and complementary pathological mechanisms to explain why lysosomal enzyme mutations lead to α-synuclein accumulation and become important risk factors in Parkinson's disease etiology. Classically, loss of GBA activity has been linked to a dysfunctional autophagy-lysosome system and to a subsequent decrease in autophagy-dependent α-synuclein turnover; however, several other pathological mechanisms underlying GBA-associated parkinsonism have been proposed. This review summarizes and discusses the different hypotheses with a special focus on autophagy-dependent mechanisms, as well as autophagy-independent mechanisms, where the role of other players such as sphingolipids, cholesterol and other GBA-related proteins make important contributions to Parkinson's disease pathogenesis.


Assuntos
Doença de Gaucher , Doença de Parkinson , Humanos , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Glucosilceramidase/metabolismo , Hidrolases , Lisossomos/metabolismo , Autofagia
6.
Arch Biochem Biophys ; 733: 109471, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36522814

RESUMO

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO2-) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH3, and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.


Assuntos
Frutose-Bifosfato Aldolase , Bases de Schiff , Frutose-Bifosfato Aldolase/genética , Cinética , Bases de Schiff/química , Bases de Schiff/metabolismo , Lisina , Mutagênicos , Sítios de Ligação , Aldeído Liases/química , Catálise , Hidrolases/metabolismo , Naftalenos , Especificidade por Substrato
7.
Protein Sci ; 32(1): e4540, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36502283

RESUMO

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l-2-haloacid dehalogenase (l-2-HAD) from the marine Bacteroidetes Zobellia galactanivorans DsijT (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l-2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l-2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l-2-HAD.


Assuntos
Flavobacterium , Hidrolases , Flavobacterium/genética , Flavobacterium/metabolismo , Raios X , Hidrolases/química , Aminoácidos , Especificidade por Substrato
8.
Bioresour Technol ; 370: 128547, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36584720

RESUMO

Hydrothermal treatment (HTT) (i.e., hydrothermal carbonization, liquefaction, and gasification) is a promising technology for biomass valorization. However, diverse variables, including biomass compositions and hydrothermal processes parameters, have impeded in-depth mechanistic understanding on the reaction and engineering in HTT. Recently, machine learning (ML) has been widely employed to predict and optimize the production of biofuels, chemicals, and materials from HTT by feeding experimental data. This review comprehensively analyzed the application of ML for HTT of biomass and systematically illustrated basic ML procedure and descriptors for inputs and outputs of ML models (e.g., biomass compositions, operation conditions, yield and physicochemical properties of derived products) that could be applied in HTT. Moreover, this review summarized ML-aided HTT prediction of yield, compositions, and physicochemical properties of HTT hydrochar or biochar, bio-oil, syngas, and aqueous phase. Ultimately, future prospects were proposed to enhance predictive performance, mechanistic interpretation, process optimization, data sharing, and model application during ML-aided HTT.


Assuntos
Biocombustíveis , Água , Temperatura , Biomassa , Hidrolases
9.
Gene ; 854: 147123, 2023 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-36535460

RESUMO

The peptidylarginine-deiminase 4 (PADI4) is involved in the post-translational catalytic conversion of arginine into citrulline. The autoantibodies including anti-citrullinated protein antibodies (ACPAs) produced in response to hypercitrullinated proteins are a hallmark of rheumatoid arthritis (RA) autoimmunity. Therefore, the role of a missense variant rs874881 (Gly112Ala) of PADI4 in RA susceptibility was analyzed, along with in-silico analysis of structural and functional impacts of this substitution. We did a case-control association study and in-silico analysis. For the case-control study, confirmed RA cases and healthy controls were recruited. Genotyping for rs874881 (n = 750) was performed through polymerase chain reaction-restriction fragment length polymorphism. Multivariate logistic regression analysis was employed to determine association. The in-silico analysis was carried out through HOPE, VarMap, MutationAssessor, MutPred2, SIFT, PolyPhen, CADD, REVEL and MetaLR. In the case-control study, the rs874881 exhibited a strong association with increased RA susceptibility (G vs C odds ratio = 3.85, 95 % confidence interval = 2.81-5.27). Interaction analysis revealed significant interaction of genotype with smoking and gender (p < 0.05). Significant results (p < 0.05) were also obtained in stratified analysis by presence/absence of comorbidities and radiographic damage. According to in-silico pathogenicity prediction analysis, this Gly112Ala substitution does not exert a major effect on protein structure and function including its enzymatic activity. We report a significant association of PADI4 rs874881 with overall RA susceptibility. To our knowledge, this is the first study to do the interaction and stratified analyses on the PADI4 rs874881 in RA. Similar detailed studies should also be performed in other populations.


Assuntos
Artrite Reumatoide , Hidrolases , Humanos , Artrite Reumatoide/genética , Estudos de Casos e Controles , Estudos de Associação Genética , Predisposição Genética para Doença , Hidrolases/genética , Polimorfismo de Nucleotídeo Único , Proteína-Arginina Desiminase do Tipo 4/genética , Desiminases de Arginina em Proteínas/genética
10.
Cell Biochem Funct ; 41(1): 128-137, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36515301

RESUMO

Dysfunction of the adipose tissue metabolism is considered as a significant hallmark of aging. It has been proposed that α-ß hydrolase domain containing 5 (ABHD5) plays a critical role in the control of lipolysis. However, the role of ABHD5 in the control of lipolysis during aging or exercise is unknown. Here we combined the experimental mouse model with transcriptomic analyzes by using murine and human databases to explore the role of ABHD5 in the adipose tissue during aging and in response to exercise. Transcriptomic data revealed a downregulation of Abhd5 messenger RNA levels in the subcutaneous white adipose tissue (scWAT) over time in individuals from 20 to 69 years old. Aged mice displayed dramatic reduction of ABHD5 protein content and lipolytic-related proteins in the scWAT. Interestingly, 4 weeks of high-intensity interval training increased ABHD5 protein level and restored the lipolytic pathway in the scWAT of aged mice. Altogether, our findings demonstrated that aging affects ABHD5 content in the adipose tissue of mice and humans. Conversely, exercise increases ABHD5 activity, recovering the lipolytic activity in aged mice.


Assuntos
Tecido Adiposo , Hidrolases , Animais , Camundongos , Humanos , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Hidrolases/genética , Hidrolases/metabolismo , Tecido Adiposo/metabolismo , Proteínas/metabolismo , Lipólise/genética , Envelhecimento , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo
11.
Methods Mol Biol ; 2609: 111-132, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36515833

RESUMO

ADP-ribosylation is an ancient modification of proteins, nucleic acids, and other biomolecules found in all kingdoms of life as well as in certain viruses. The regulation of fundamental (patho)physiological processes by ADP-ribosylation, including the cellular stress response, inflammation, and immune response to bacterial and viral pathogens, has created a strong interest into the study of modification establishment and removal to explore novel therapeutic approaches. Beyond ADP-ribosylation in humans, direct targeting of factors that alter host ADP-ribosylation signaling (e.g., viral macrodomains) or utilize ADP-ribosylation to manipulate host cell behavior (e.g., bacterial toxins) were shown to reduce virulence and disease severity. However, the realization of these therapeutic potentials is thus far hampered by the unavailability of simple, high-throughput methods to study the modification "writers" and "erasers" and screen for novel inhibitors.Here, we describe a scalable method for the measurement of (ADP-ribosyl)hydrolase activity. The assay relies on the conversion of ADP-ribose released from a modified substrate by the (ADP-ribosyl)hydrolase under investigation into AMP by the phosphodiesterase NudT5 into bioluminescence via a commercially available detection assay. Moreover, this method can be utilized to study the role of nudix- or ENPP-type phosphodiesterases in ADP-ribosylation processing and may also be adapted to investigate the activity of (ADP-ribosyl)transferases. Overall, this method is applicable for both basic biochemical characterization and screening of large drug libraries; hence, it is highly adaptable to diverse project needs.


Assuntos
ADP-Ribosilação , Adenosina Difosfato Ribose , Humanos , Adenosina Difosfato Ribose/química , Proteínas/química , Diester Fosfórico Hidrolases/metabolismo , Hidrolases/metabolismo , Descoberta de Drogas
12.
Chemosphere ; 314: 137673, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36584821

RESUMO

Multi-pesticides pollution induced by organophosphorus insecticides (OPs) and aryloxyphenoxypropionate herbicides (AOPPs) has become a significant challenge in bioremediation of water pollution due to their prolonged and over application. Though a number of physical, chemical, and biological approaches have been developed for different pesticides, the explorations usually focus on eliminating single pesticide pollution. Herein, a heterostructure nanocomposite OPH/QpeH@mZIF-8, encapsulating OPs hydrolase OPH and AOPPs hydrolase QpeH in the magnetic zeolitic imidazolate frameworks-8 (mZIF-8), was synthesized through a facile one-pot method in aqueous solution. The immobilized OPH and QpeH in mZIF-8 showed high activities towards the two most common OPs and AOPPs, i.e., chlorpyrifos and quizalofop-P-ethyl, which were hydrolyzed to 3,5,6-Trichloro-2-pyridino (TCP) and quizalofop acid, respectively. Moreover, the magnetic nanocatalyst possessed great tolerance towards broad pH range, high temperatures, and different chemical solvents and excellent recyclability. More importantly, compared to free OPH and QpeH, OPH/QpeH@mZIF-8, with significantly enhanced degradation capability, exhibited enormous potential for simultaneous removal of chlorpyrifos and quizalofop-p-ethyl from the surface and industrial wastewater. Overall, the study demonstrates the applicability of this strategy for utilizing magnetic nanocatalysts encapsulating multiple enzymes due to its simplicity, high efficiency, and economic benefits to removing pesticide compound pollution from various water resources.


Assuntos
Clorpirifos , Herbicidas , Praguicidas , Praguicidas/análise , Hidrolases/metabolismo , Água , Produtos da Oxidação Avançada de Proteínas , Fenômenos Magnéticos
13.
J Med Chem ; 66(1): 538-552, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36516997

RESUMO

Multimodal imaging provides rich biological information, which can be exploited to study drug activity, disease associated phenotypes, and pharmacological responses. Here we show discovery and validation of a new probe targeting the endocannabinoid α/ß-hydrolase domain 6 (ABHD6) enzyme by utilizing positron emission tomography (PET) and matrix-assisted laser desorption/ionization (MALDI) imaging. [18F]JZP-MA-11 as the first PET ligand for in vivo imaging of the ABHD6 is reported and specific uptake in ABHD6-rich peripheral tissues and major brain regions was demonstrated using PET. A proof-of-concept study in nonhuman primate confirmed brain uptake. In vivo pharmacological response upon ABHD6 inhibition was observed by MALDI imaging. These synergistic imaging efforts used to identify biological information cannot be obtained by a single imaging modality and hold promise for improving the understanding of ABHD6-mediated endocannabinoid metabolism in peripheral and central nervous system disorders.


Assuntos
Endocanabinoides , Hidrolases , Animais , Endocanabinoides/metabolismo , Hidrolases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Monoacilglicerol Lipases , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Tomografia por Emissão de Pósitrons
14.
J Mol Graph Model ; 119: 108379, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36481587

RESUMO

The binding affinity of the SARS-CoV-2 spike (S)-protein to the human membrane protein ACE2 is critical for virus function. Computational structure-based screening of new S-protein mutations for ACE2 binding lends promise to rationalize virus function directly from protein structure and ideally aid early detection of potentially concerning variants. We used a computational protocol based on cryo-electron microscopy structures of the S-protein to estimate the change in ACE2-affinity due to S-protein mutation (ΔΔGbind) in good trend agreement with experimental ACE2 affinities. We then expanded predictions to all possible S-protein mutations in 21 different S-protein-ACE2 complexes (400,000 ΔΔGbind data points in total), using mutation group comparisons to reduce systematic errors. The results suggest that mutations that have arisen in major variants as a group maintain ACE2 affinity significantly more than random mutations in the total protein, at the interface, and at evolvable sites. Omicron mutations as a group had a modest change in binding affinity compared to mutations in other major variants. The single-mutation effects seem consistent with ACE2 binding being optimized and maintained in omicron, despite increased importance of other selection pressures (antigenic drift), however, epistasis, glycosylation and in vivo conditions will modulate these effects. Computational prediction of SARS-CoV-2 evolution remains far from achieved, but the feasibility of large-scale computation is substantially aided by using many structures and mutation groups rather than single mutation effects, which are very uncertain. Our results demonstrate substantial challenges but indicate ways forward to improve the quality of computer models for assessing SARS-CoV-2 mutation effects.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Humanos , Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Microscopia Crioeletrônica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Hidrolases , Mutação , Ligação Proteica
15.
Biochemistry ; 62(2): 524-534, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36563174

RESUMO

2,4-Diketo-3-deoxy-l-rhamnonate (L-DKDR) hydrolase (LRA6) catalyzes the hydrolysis reaction of L-DKDR to pyruvate and l-lactate in the nonphosphorylated l-rhamnose pathway from bacteria and belongs to the fumarylacetoacetate hydrolase (FAH) superfamily. Most of the members of the FAH superfamily are involved in the microbial degradation of aromatic substances and share low sequence similarities with LRA6, by which the underlying catalytic mechanism remains unknown at the atomic level. We herein elucidated for the first time the crystal structures of LRA6 from Sphingomonas sp. without a ligand and in complex with pyruvate, in which a magnesium ion was coordinated with three acidic residues in the catalytic center. Structural, biochemical, and phylogenetic analyses suggested that LRA6 is a close but distinct subfamily of the fumarylpyruvate hydrolase (FPH) subfamily, and amino acid residues at equivalent position to 84 in LRA6 are related to different substrate specificities between them (Leu84 and Arg86 in LRA6 and FPH, respectively). Structural transition induced upon the binding of pyruvate was observed within a lid-like region, by which a glutamate-histidine dyad that is critical for catalysis was arranged sufficiently close to the ligand. Among several hydroxylpyruvates (2,4-diketo-5-hydroxycarboxylates), L-DKDR with a C6 methyl group was the best substrate for LRA6, conforming to the physiological role. Significant activity was also detected in acylpyruvate including acetylpyruvate. The structural analysis presented herein provides a more detailed understanding of the molecular evolution and physiological role of the FAH superfamily enzymes (e.g., the FAH like-enzyme involved in the mammalian l-fucose pathway).


Assuntos
Hidrolases , Ramnose , Animais , Ramnose/metabolismo , Filogenia , Ligantes , Hidrolases/química , Bactérias/metabolismo , Piruvatos , Cristalografia por Raios X , Mamíferos/metabolismo
16.
J Am Chem Soc ; 145(2): 1083-1096, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36583539

RESUMO

Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91's activity by a factor of ≈360 (to a kcat/KM of ≈7 × 105 M-1 s-1) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.


Assuntos
Hidrolases , Hidrolases de Triester Fosfórico , Acetilcolinesterase , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Biocatálise , Catálise
17.
J Cell Physiol ; 237(1): 1044-1056, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34553380

RESUMO

Kynureninase (KYNU) is a key enzyme in the tryptophan metabolism pathway with elevated expression in psoriatic lesions relative to normal skin. However, whether KYNU contributes to the pathogenesis of psoriasis remains unknown. We sought to investigate the role of KYNU in psoriasis and its possible regulation mechanism. In the results, KYNU is upregulated in psoriatic skin samples from patients or animal models compared with normal skin control which was assayed in psoriatic patient samples, IMQ-induced psoriasis-like skin inflammation in BABL/c mice and M5-stimulated keratinocyte cell lines by immunohistochemistry (IHC). KYNU knockdown had a trivial impact on keratinocyte proliferation, but significantly inhibited the production of inflammatory cytokines in HaCaT, HEKα, and HEKn cells by quantitative reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and western blot analysis. The 3'-untranslated region of KYNU contains a conserved target site of a skin-specific microRNA (miRNA), miR-203a, as predicted by TargetScan software. Furthermore, miR-203a exhibited an inversed expression kinetics to KYNU during the development of IMQ-induced psoriasis-like skin inflammation in BABL/c mice. Overexpression of miR-203 subsequently leading to the inhibition of KYNU, could significantly reduce the production of M5-induced, psoriasis-related inflammatory factors in keratinocytes. Finally, KYNU inhibitors could alleviate the pathological phenotypes in IMQ-mice. Our study supported the contributive role of KYNU in the development of psoriasis and provided preliminary evidence for KYNU as a potential therapeutic target in psoriasis.


Assuntos
MicroRNAs , Psoríase , Animais , Proliferação de Células/genética , Humanos , Hidrolases , Imiquimode/efeitos adversos , Inflamação/metabolismo , Queratinócitos/metabolismo , Camundongos , MicroRNAs/metabolismo , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Psoríase/genética , Pele/metabolismo
18.
Food Res Int ; 162(Pt B): 112092, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36461399

RESUMO

Gastrodiae Rhizoma is a commonly used plant material for both medicine and food in China for thousands of years. Steaming as the main pre-processing method of Gastrodiae Rhizoma is the key to its quality formation. In this study, we established a high-coverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) method to visualize the spatial distribution of phenols in Gastrodiae Rhizoma. By optimizing 1,5-diaminonaphthalene as MALDI matrix in negative mode, the "spatial-temporal-content" changes of 13 phenols including 11 parishins in Gastrodiae Rhizoma during the steaming process were imaged. We also characterized the activity of two hydrolases related to parishins degradation during the steaming. Moreover, the degradation pathways of parishins in Gastrodiae Rhizoma were summarized. The integration of spatially-resolved parishins and corresponding hydrolases information revealed the scientific connotation of steaming as the processing method of Gastrodiae Rhizoma is to inactivate hydrolases and protect parishins, which provided theoretical support for the quality improvement and the standardized processing of Gastrodiae Rhizoma.


Assuntos
Rizoma , Vapor , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Hidrolases , Fenóis , Lasers
19.
Viruses ; 14(12)2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36560748

RESUMO

Chikungunya virus (CHIKV) causes outbreaks of rash, arthritis, and fever associated with neurologic complications, where astrocytes are preferentially infected. A determinant of virulence is the macrodomain (MD) of nonstructural protein 3 (nsP3), which binds and removes ADP-ribose (ADPr) from ADP-ribosylated substrates and regulates stress-granule disruption. We compared the replication of CHIKV 181/25 (WT) and MD mutants with decreased ADPr binding and hydrolase (G32S) or increased ADPr binding and decreased hydrolase (Y114A) activities in C8-D1A astrocytic cells and NSC-34 neuronal cells. WT CHIKV replication was initiated more rapidly with earlier nsP synthesis in C8-D1A than in NSC-34 cells. G32S established infection, amplified replication complexes, and induced host-protein synthesis shut-off less efficiently than WT and produced less infectious virus, while Y114A replication was close to WT. However, G32S mutation effects on structural protein synthesis were cell-type-dependent. In NSC-34 cells, E2 synthesis was decreased compared to WT, while in C8-D1A cells synthesis was increased. Excess E2 produced by G32S-infected C8-D1A cells was assembled into virus particles that were less infectious than those from WT or Y114A-infected cells. Because nsP3 recruits ADP-ribosylated RNA-binding proteins in stress granules away from translation-initiation factors into nsP3 granules where the MD hydrolase can remove ADPr, we postulate that suboptimal translation-factor release decreased structural protein synthesis in NSC-34 cells while failure to de-ADP-ribosylate regulatory RNA-binding proteins increased synthesis in C8-D1A cells.


Assuntos
Febre de Chikungunya , Vírus Chikungunya , Humanos , Proteínas não Estruturais Virais/metabolismo , Vírus Chikungunya/genética , Replicação Viral/genética , Proteínas de Ligação a RNA/metabolismo , Hidrolases , Adenosina Difosfato Ribose/metabolismo
20.
Biopharm Drug Dispos ; 43(6): 247-254, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36519186

RESUMO

As an analog of clopidogrel and prasugrel, vicagrel is completely hydrolyzed to intermediate thiolactone metabolite 2-oxo-clopidogrel (also the precursor of active thiol metabolite H4) in human intestine, predominantly by AADAC and CES2; however, other unknown vicagrel hydrolases remain to be identified. In this study, recombinant human Raf kinase inhibitor protein (rhRKIP) and pooled human intestinal S9 (HIS9) fractions and microsome (HIM) preparations were used as the different enzyme sources; prasugrel as a probe drug for RKIP (a positive control), vicagrel as a substrate drug of interest, and the rate of the formation of thiolactone metabolites 2-oxo-clopidogrel and R95913 as metrics of hydrolase activity examined, respectively. In addition, an IC50 value of inhibition of rhRKIP-catalyzed vicagrel hydrolysis by locostatin was measured, and five classical esterase inhibitors with distinct esterase selectivity were used to dissect the involvement of multiple hydrolases in vicagrel hydrolysis. The results showed that rhRKIP hydrolyzed vicagrel in vitro, with the values of Km , Vmax , and CLint measured as 20.04 ± 1.99 µM, 434.60 ± 12.46 nM/min/mg protein, and 21.69 ± 0.28 ml/min/mg protein, respectively, and that an IC50 value of locostatin was estimated as 1.24 ± 0.04 mM for rhRKIP. In addition to locostatin, eserine and vinblastine strongly suppressed vicagrel hydrolysis in HIM. It is concluded that RKIP can catalyze the hydrolysis of vicagrel in the human intestine, and that vicagrel can be hydrolyzed by multiple hydrolases, such as RKIP, AADAC, and CES2, concomitantly.


Assuntos
Hidrolases , Proteína de Ligação a Fosfatidiletanolamina , Humanos , Cloridrato de Prasugrel/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Clopidogrel , Hidrolases/metabolismo , Esterases/metabolismo , Intestinos
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