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1.
JACS Au ; 4(6): 2151-2159, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38938820

RESUMO

This report develops a point-of-use chemical trigger and applies it to a dual-functional chemical encryption chip that enables manual and digital identification with enhanced coding security levels suitable for on-site information verification. The concept relies on conducting continuous chemical synthesis and chromatographic separation of specified compounds on a paper device in a straightforward sketch. In addition to single-step chemical reactions, cascade syntheses and operations involving components of distinct mobilities are also demonstrated. The condensation of dione and hydrazine is first demonstrated on a linear paper reactor, where precursors can mix to react, followed by final product separation under optimized conditions. This linear paper reactor design can also support a multistep cascade Wittig reaction by controlling the relative mobility of reactants, intermediates, and final products. Furthermore, a three-dimensional paper reactor with appropriate mobile phases helps to initiate complex solvent system-driven azide-alkyne cycloaddition. By the use of a three-dimensional device design for spatially limited interdevice reactant transportation, reactants crossing designated boundaries trigger confined chemical reactions at specific positions. Accumulation of repetitive reactions leads to successful product gradient generation and mixing effects, representing a fully controllable intersubstrate chemical operation on the platform. Standing on initiating desired chemical reactions at particular interface regions, integration of appropriate selective reaction area, numerical digits overlay, color diversity, and mobile recognition realizes this dual-functional multicoding encryption process.

2.
Bioorg Med Chem ; 98: 117582, 2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38171253

RESUMO

In this study, we explored a concise and mild synthetic route to produce novel C-14 arylcarbamate derivatives of andrographolide, a known anti-inflammatory and anticancer natural product. Upon assessing their anti-cancer efficacy against pancreatic ductal adenocarcinoma (PDAC) cells, some derivatives showed stronger cytotoxicity against PANC-1 cells than andrographolide. In addition, we demonstrated one derivative, compound 3m, effectively reduced the expression of oncogenic p53 mutant proteins (p53R273H and p53R248W), proliferation, and migration in PDAC lines, PANC-1 and MIA PaCa-2. Accordingly, the novel derivative holds promise as an anti-cancer agent against pancreatic cancer. In summary, our study broadens the derivative library of andrographolide and develops an arylcarbamate derivative of andrographolide with promising anticancer activity against PDAC.


Assuntos
Carcinoma Ductal Pancreático , Diterpenos , Neoplasias Pancreáticas , Humanos , Proteína Supressora de Tumor p53/metabolismo , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Diterpenos/farmacologia , Linhagem Celular Tumoral
3.
RSC Adv ; 13(25): 17420-17426, 2023 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-37304775

RESUMO

A thermally stable, fluorous sulfur-containing boronic acid catalyst has been developed and was shown to efficiently promote dehydrative condensation between carboxylic acids and amines under environmentally friendly conditions. The methodology can be applied to aliphatic, aromatic and heteroaromatic acids as well as primary and secondary amines. N-Boc protected amino acids were also successfully coupled in good yields with very little racemization. The catalyst could be reused four times with no significant loss of activity.

4.
J Cell Sci ; 135(10)2022 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-35466366

RESUMO

Tripeptidyl peptidase II (TPPII or TPP2) degrades N-terminal tripeptides from proteins and peptides. Studies in both humans and mice have shown that TPPII deficiency is linked to cellular immune-senescence, lifespan regulation and the aging process. However, the mechanism of how TPPII participates in these processes is less clear. In this study, we established a chemical probe-based assay and found that although the mRNA and protein levels of TPPII were not altered during senescence, its enzymatic activity was reduced in senescent human fibroblasts. We also showed that elevation of the levels of the serine protease inhibitor serpinB2 reduced TPPII activity in senescent cells. Moreover, suppression of TPPII led to elevation in the amount of lysosomal contents as in well as TPPI (TPP1) and ß-galactosidase activities, suggesting that lysosome biogenesis is induced to compensate for the reduction of TPPII activity in senescent cells. Together, this study discloses a critical role of the serpinB2-TPPII signaling pathway in proteostasis during senescence. Since serpinB2 levels can be increased by a variety of cellular stresses, reduction of TPPII activity through activation of serpinB2 might represent a common pathway for cells to respond to different stress conditions. This article has an associated First Person interview with the first author of the paper.


Assuntos
Aminopeptidases , Senescência Celular , Dipeptidil Peptidases e Tripeptidil Peptidases , Peptídeos e Proteínas de Sinalização Intracelular , Aminopeptidases/genética , Aminopeptidases/metabolismo , Senescência Celular/genética , Senescência Celular/fisiologia , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteostase/genética , Proteostase/fisiologia , Serina Endopeptidases/metabolismo , Transdução de Sinais
5.
J Virol ; 96(7): e0010722, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35293767

RESUMO

The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N6-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.


Assuntos
Hepacivirus , Hepatite C , RNA Polimerase Dependente de RNA , Proteínas não Estruturais Virais , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Caseína Quinase Ialfa/metabolismo , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C/virologia , Humanos , Fosforilação , Poliproteínas/metabolismo , Domínios Proteicos/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
6.
Biochem Biophys Res Commun ; 533(3): 467-473, 2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-32977949

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (Mpro) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys145 of either 2019-nCoV Mpro or SARS-CoV Mpro. Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral Mpros. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested.


Assuntos
Cisteína Endopeptidases/metabolismo , Diterpenos/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Betacoronavirus/enzimologia , Domínio Catalítico , Proteases 3C de Coronavírus , Cisteína Endopeptidases/química , Diterpenos/química , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Simulação de Acoplamento Molecular , Conformação Proteica , Multimerização Proteica , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , SARS-CoV-2 , Proteínas não Estruturais Virais/química
7.
ACS Comb Sci ; 22(11): 600-607, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32833425

RESUMO

The preparation of natural product-inspired nucleoside analogs using solution-phase parallel synthesis is described. The key intermediates containing alkyne and N-protected amino moieties were developed to allow for further skeleton and substituent diversity using click chemistry and urea or amide bond formation. Rapid purification was accomplished using solid-phase extraction. The obtained library comprised 80 molecules incorporating two diversity positions and one chiral center, each of which was efficiently prepared in good purity and acceptable overall yield. A bacterial morphology study was also performed.


Assuntos
Produtos Biológicos/síntese química , Nucleosídeos/química , Bibliotecas de Moléculas Pequenas/síntese química , Uridina/química , Alcinos/química , Amidas/química , Bacillus subtilis , Química Click , Técnicas de Química Combinatória , Estrutura Molecular , Pró-Fármacos/química , Estereoisomerismo , Ureia/química
8.
PLoS One ; 11(4): e0152770, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27035713

RESUMO

Andrographolide (ANDRO) is a lactone diterpenoid compound present in the medicinal plant Andrographis paniculata which is clinically applied for multiple human diseases in Asia and Europe. The pharmacological activities of andrographolide have been widely demonstrated, including anti-inflammation, anti-cancer and hepatoprotection. However, the pharmacological mechanism of andrographolide remains unclear. Therefore, further characterization on the kinetics and molecular targets of andrographolide is essential. In this study, we described the synthesis and characterization of a novel fluorescent andrographolide derivative (ANDRO-NBD). ANDRO-NBD exhibited a comparable anti-cancer spectrum to andrographolide: ANDRO-NBD was cytotoxic to various types of cancer cells and suppressed the migration activity of melanoma cells; ANDRO-NBD treatment induced the cleavage of heat shock protein 90 (Hsp90) and the downregulation of its client oncoproteins, v-Src and Bcr-abl. Notably, ANDRO-NBD showed superior inhibitory effects to andrographolide in all anticancer assays we have performed. In addition, ANDRO-NBD was further used as a fluorescent probe to investigate the uptake kinetics, cellular distribution and molecular targets of andrographolide. Our data revealed that ANDRO-NBD entered cells rapidly and its fluorescent signal could be detected in nucleus, cytoplasm, mitochondria, and lysosome. Moreover, we demonstrated that ANDRO-NBD was covalently bound to several putative target proteins of andrographolide, including NF-κB and hnRNPK. In summary, we developed a fluorescent andrographolide probe with comparable bioactivity to andrographolide, which serves as a powerful tool to explore the pharmacological mechanism of andrographolide.


Assuntos
Diterpenos/química , Sondas Moleculares/química , Adulto , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade
9.
J Org Chem ; 80(16): 8458-63, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26240938

RESUMO

We have established a concise synthetic route relying on a key base-promoted epimerization step to synthesize two series of activity-based probes carrying a BODIPY fluorophore for α-l-fucosidase. The resulting probes were evaluated for labeling performance. The one utilizing an o-fluoromethylphenol derivative as the latent trapping unit was successfully applied for the first time to visualize and locate lysosomal α-l-fucosidase activity in human cells.


Assuntos
Compostos de Boro/química , Membrana Celular/química , Fenóis/química , alfa-L-Fucosidase/química , Membrana Celular/enzimologia , Fluorescência , Humanos , Cinética
10.
Chembiochem ; 16(11): 1555-9, 2015 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-26096673

RESUMO

α-L-Fucosidase activity is associated with several diseases. To study the enzymatic activity change under pathological conditions, we developed a quinone methide-generating activity-based probe useful for examining the presence, activity, and localization of human α-L-fucosidase in vivo in the context of Helicobacter pylori infection. In particular, an increase in intracellular fucosidase (Fuca1) activity was found in gastric epithelial cells upon bacterial infection. We further studied the effect of several bacterial stimulants on this enhanced Fuca1 activity and identified lipopolysaccharides to be a major contributing factor.


Assuntos
Helicobacter pylori/fisiologia , Indolquinonas/metabolismo , Sondas Moleculares/metabolismo , alfa-L-Fucosidase/metabolismo , Linhagem Celular Tumoral , Epitélio/microbiologia , Humanos , Lipopolissacarídeos/metabolismo , Estômago/microbiologia
11.
Chem Commun (Camb) ; 50(46): 6116-9, 2014 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-24776788

RESUMO

We have synthesized and evaluated two self-immobilizing, turn-on fluorescent probes carrying a coumarin molecular framework for imaging intracellular human steroid sulfatase (STS) activity. The 8-fluoromethyl coumarin derivative, which gives stronger fluorescence response in the incubation study with STS preparations, was successfully applied to visualize STS activity in cells.


Assuntos
Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Esteril-Sulfatase/metabolismo , Linhagem Celular , Ativação Enzimática , Humanos , Estrutura Molecular , Esteril-Sulfatase/análise
12.
J Med Chem ; 56(7): 2841-9, 2013 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-23470190

RESUMO

Specific interactions between Src homology 2 (SH2) domain-containing proteins and the phosphotyrosine-containing counterparts play significant role in cellular protein tyrosine kinase (PTK) signaling pathways. The SH2 domain inhibitors could potentially serve as drug candidates in treating human diseases. Here we have incorporated a novel phosphotyrosine mimetic, which is an unusual amino acid carrying a cyclosaligenyl (cycloSal) phosphodiester moiety, into dipeptides to investigate the inhibitory effect on SH2 domain-containing proteins. A plate-based assay was also established to screen for inhibitors that disrupt the interaction between a phosphopeptide of SLAM (signaling lymphocytic activation molecule) and its interacting protein SAP (SLAM-associated protein). We identified a number of inhibitors with IC50 values in the range of 17-35 µM, implying that the cycloSal phosphodiester-carrying amino acid could mimic the phosphotyrosyl residue. Our results also raise the possibility of integrating the newly developed phosphotyrosine mimetic moiety into inhibitors designed for other SH2 domain-containing proteins.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Mimetismo Molecular , Fosfotirosina/farmacologia , Domínios de Homologia de src , Sítios de Ligação , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária
13.
Molecules ; 18(1): 682-9, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23292330

RESUMO

Two new diterpenoids, konishone (1) and 3b-hydroxy-5,6-dehydrosugiol (2), along with three known diterpenoids--hinokiol (3), sugiol (4), and 12-hydroxy-6,7-secoabieta-8,11,13-triene-6,7-dial (5)--were isolated from the wood of Cunninghamia konishii. Compound 1 is a novel skeleton of the 7,20-dinorabietane-type diterpene. In addition, when RAW264.7 macrophages were treated with different concentrations of compounds 1, 3, and 5 together with LPS, a significant concentration-dependent inhibition of NO production was detected. The IC50 values for inhibition of nitrite production of compounds 1, 3, and 5 were about 9.8 ± 0.7, 7.9 ± 0.9, and 9.3 ± 1.3 µg/mL, respectively. This study presents the potential utilization of compounds 1, 3, and 5, as lead compounds for the development of anti-inflammatory drugs.


Assuntos
Anti-Inflamatórios/farmacologia , Cunninghamia/química , Diterpenos/farmacologia , Madeira/química , Animais , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Diterpenos/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Óxido Nítrico/metabolismo
14.
J Org Chem ; 77(6): 2729-42, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22372634

RESUMO

To facilitate precatalyst recovery and reuse, we have developed a fluorous, oxime-based palladacycle 1 and demonstrated that it is a very efficient and versatile precatalyst for a wide range of carbon-carbon bond formation reactions (Suzuki-Miyaura, Sonogashira, Stille, Heck, Glaser-type, and Kumada) in either aqueous or organic medium under microwave irradiation. Palladacycle 1 could be recovered through F-SPE in various coupling reactions with recovery ranging from 84 to 95% for the first cycle. Inductively coupled plasma optical emission spectrometry (ICP-OES) analyses of the Pd content in the crude product from each class of transformation indicated extremely low levels of leaching and the palladacycle could be reused four to five times without significant loss of activity.

15.
J Agric Food Chem ; 59(21): 11403-6, 2011 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-21967291

RESUMO

Hydrogen peroxide is commonly used in the food processing industry as a chlorine-free bleaching and sterilizing agent, but excessive amounts of residual hydrogen peroxide have led to cases of food poisoning. Here we describe the development of a novel nonenzymatic colorimetric method for the determination of residual hydrogen peroxide in foods and agricultural products. Nitrophenylboronic acids chemoselectively react with hydrogen peroxide under alkaline conditions to produce yellow nitrophenolates. Of the three nitrophenylboronic acid isomers tested, the p-isomer displayed the highest sensitivity for hydrogen peroxide and the fastest reaction kinetics. The reaction product, p-nitrophenolate, has an absorption maximum at 405 nm and a good linear correlation between the hydrogen peroxide concentration and the A(405) values was obtained. We successfully applied this convenient and rapid method for hydrogen peroxide determination to samples of dried bean curds and disposable chopsticks, thereby demonstrating its potential in foods and agricultural industries.


Assuntos
Ácidos Borônicos/química , Colorimetria/métodos , Análise de Alimentos/métodos , Conservantes de Alimentos/análise , Peróxido de Hidrogênio/análise , Sensibilidade e Especificidade
16.
Biochem Biophys Res Commun ; 391(1): 230-4, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19909727

RESUMO

Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.


Assuntos
Antineoplásicos/farmacologia , Biotina/análogos & derivados , Cisplatino/farmacologia , Sondas Moleculares/química , Neoplasias/enzimologia , Organofosfatos/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/biossíntese , Anticorpos/imunologia , Bioensaio , Biotina/química , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Proteínas Tirosina Fosfatases/metabolismo
18.
Biochem J ; 401(2): 551-8, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17002602

RESUMO

A GH (glycoside hydrolase) family 54 alpha-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii alpha-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-alpha-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, beta(lg)=-0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pK(a) values (>6) of leaving phenols, with beta(lg)=-1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Trichoderma/enzimologia , Sequência de Aminoácidos , Arabinose/análogos & derivados , Arabinose/síntese química , Sítios de Ligação , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular
19.
Biotechnol J ; 1(2): 197-202, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16892248

RESUMO

beta-Xylosidase is a key enzyme in the xylanolytic system with a great potential in many biotechnological applications, especially in the food as well as the pulp and paper industries. We have developed a chemical approach for the rapid screening and isolation of beta-xylosidase. Activity probe LCL-6X targeting beta-xylosidase was utilized in this study. It carries a beta-xylopyranosyl recognition head, a latent trapping device consisting of a 2-fluoromethylphenoxyl group, and a biotin reporter group. The biotin reporter group serves both as a readout device and as a tool for enriching the labeled proteins. LCL-6X could selectively label a model beta-xylosidase from Trichoderma koningii. All other bystander proteins used in this study, including phosphorylase b, BSA, ovalbumin, carbonic anhydrase, and trypsin inhibitor, gave negligible cross-labeling effect. With the assistance of streptavidin agarose beads and mass spectrophotometry for the recovery and identification of the biotinylated proteins, we demonstrated that LCL-6X could be successfully applied to identify a bi-functional enzyme with alpha-L-arabinofuranosidase/beta-xylosidase activity from the total protein extract of a Pichia expressing system and a prospective beta-xylosidase in the culture medium of Aspergillus fumigatus. The beta-xylosidase activities from numerous microbes were also screened using the LCL-6X probe. Preliminary results showed significant differences among these microbial sources and some distinct protein bands were observed. Thus, we have successfully developed a novel chemical probe that has potential applications in xylan-related research.


Assuntos
Fracionamento Químico/métodos , Trichoderma/enzimologia , Xilosidases/química , Xilosidases/isolamento & purificação , Ativação Enzimática , Técnicas de Sonda Molecular , Sensibilidade e Especificidade
20.
Carbohydr Res ; 341(4): 443-56, 2006 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-16414035

RESUMO

Chemical probes that selectively label the glycoside hydrolase (GH) subfamilies have proven to be a powerful tool in GH-related research. We have previously demonstrated the design and synthesis of an activity probe for beta-glucosidase adopting a cassette-like design in a model study. Herein we report an improved synthetic route using (4-hydroxyphenyl)acetic acid 2-cyanoethyl ester as the precursor for the latent trapping device. Parallel syntheses were performed for the preparation of a library based on the structure of a key intermediate. The recognition head of this library covers a series of six sugars, including alpha- and beta-d-Glc, alpha- and beta-d-Gal, alpha-d-Man, and alpha-l-Fuc. Each member in this versatile intermediate library could serve as the building block in constructing an activity probe for GHs. As demonstrated in this study, three probes that have the 1,2-cis configuration were thus prepared for the first time to target alpha-d-glucosidase, alpha-d-galactosidase, and alpha-l-fucosidase, respectively.


Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/síntese química , Sondas Moleculares/química , Sondas Moleculares/síntese química , Técnicas de Química Combinatória , Estrutura Molecular , Proteômica
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