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1.
Nucleic Acids Res ; 51(14): 7675-7690, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-37377432

RESUMO

Reprogramming cellular behaviour is one of the hallmarks of synthetic biology. To this end, prokaryotic allosteric transcription factors (aTF) have been repurposed as versatile tools for processing small molecule signals into cellular responses. Expanding the toolbox of aTFs that recognize new inducer molecules is of considerable interest in many applications. Here, we first establish a resorcinol responsive aTF-based biosensor in Escherichia coli using the TetR-family repressor RolR from Corynebacterium glutamicum. We then perform an iterative walk along the fitness landscape of RolR to identify new inducer specificities, namely catechol, methyl catechol, caffeic acid, protocatechuate, L-DOPA, and the tumour biomarker homovanillic acid. Finally, we demonstrate the versatility of these engineered aTFs by transplanting them into the model eukaryote Saccharomyces cerevisiae. This work provides a framework for efficient aTF engineering to expand ligand specificity towards novel molecules on laboratory timescales, which, more broadly, is invaluable across a wide range of applications such as protein and metabolic engineering, as well as point-of-care diagnostics.


Assuntos
Corynebacterium glutamicum , Proteínas de Escherichia coli , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo
2.
J Biol Chem ; 299(3): 102931, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36682498

RESUMO

The sugar moieties of many glycosylated small molecule natural products are essential for their biological activity. Glycosyltransferases (GTs) are enzymes responsible for installing these sugar moieties on a variety of biomolecules. Many GTs active on natural products are inherently substrate promiscuous and thus serve as useful tools in manipulating natural product glycosylation to generate new combinations of sugar units (glycones) and scaffold molecules (aglycones) in a process called glycodiversification. It is important to have an effective screening tool to detect the activity of promiscuous enzymes and their resulting glycoside products. Toward this aim, we developed a strategy for screening natural product GTs in a high-throughput fashion enabled by rapid isolation and detection of chromophoric or fluorescent glycosylated natural products. This involves a solvent extraction step to isolate the resulting polar glycoside product from the unreacted aglycone acceptor substrate and the detection of the formed glycoside by the innate absorbance or fluorescence of the aglycone moiety. Using our approach, we screened a collection of natural product GTs against a panel of precursors to therapeutically important molecules. Three GTs showed previously unreported promiscuity toward anthraquinones resulting in novel ε-rhodomycinone glycosides. Considering the pharmaceutical value of clinically used anthraquinone glycosides that are biosynthesized from an ε-rhodomycinone precursor, and the significance that the sugar moiety has on the biological activity of these drugs, our results are of particular importance toward the glycodiversification of therapeutics in this class. The GTs identified and the novel compounds they produce show promise toward new biocatalytic tools and therapeutics.


Assuntos
Produtos Biológicos , Descoberta de Drogas , Glicosídeos , Glicosiltransferases , Antraquinonas/química , Produtos Biológicos/química , Glicosídeos/síntese química , Glicosídeos/isolamento & purificação , Glicosiltransferases/metabolismo , Açúcares , Ensaios de Triagem em Larga Escala , Descoberta de Drogas/métodos
3.
Molecules ; 26(2)2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33450899

RESUMO

This review provides information on available methods for engineering glycan-binding proteins (GBP). Glycans are involved in a variety of physiological functions and are found in all domains of life and viruses. Due to their wide range of functions, GBPs have been developed with diagnostic, therapeutic, and biotechnological applications. The development of GBPs has traditionally been hindered by a lack of available glycan targets and sensitive and selective protein scaffolds; however, recent advances in glycobiology have largely overcome these challenges. Here we provide information on how to approach the design of novel "designer" GBPs, starting from the protein scaffold to the mutagenesis methods, selection, and characterization of the GBPs.


Assuntos
Anticorpos/química , Lectinas/química , Polissacarídeos/química , Engenharia de Proteínas , Receptores de Superfície Celular/química , Sítios de Ligação
4.
Biochemistry ; 59(22): 2100-2110, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32441090

RESUMO

We report a straightforward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of N-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequentially coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.


Assuntos
Corantes Fluorescentes/metabolismo , Fucose/metabolismo , Fucosiltransferases/metabolismo , Glicosídeos/biossíntese , Ensaios de Triagem em Larga Escala , Corantes Fluorescentes/química , Fucose/química , Glicosídeos/química , Humanos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo
5.
Angew Chem Int Ed Engl ; 58(6): 1632-1637, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30549167

RESUMO

Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3-, 4-, and 6-thiosugar acceptors from a variety of sugar families produces S-linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine-containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.


Assuntos
Acetilglucosamina/química , Peptídeos/química , Proteínas/química , Açúcares/química , Compostos de Sulfidrila/química , beta-N-Acetil-Hexosaminidases/química , Acetilglucosamina/metabolismo , Estrutura Molecular , Mutação , Peptídeos/metabolismo , Proteínas/metabolismo , Streptomyces/enzimologia , Açúcares/metabolismo , Compostos de Sulfidrila/metabolismo , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismo
6.
Glycobiology ; 25(8): 806-11, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25964111

RESUMO

A facile enzymatic synthesis of the methylumbelliferyl ß-glycoside of the type 2 A blood group tetrasaccharide in good yields is reported. Using this compound, we developed highly sensitive fluorescence-based high-throughput assays for both endo-ß-galactosidase and α-N-acetylgalactosaminidase activity specific for the oligosaccharide structure of the blood group A antigen. We further demonstrate the potential to use this assay to screen the expressed gene products of metagenomic libraries in the search for efficient blood group antigen-cleaving enzymes.


Assuntos
Sistema ABO de Grupos Sanguíneos/química , Glicosídeos/síntese química , Himecromona/síntese química , Oligossacarídeos/síntese química , alfa-N-Acetilgalactosaminidase/química , beta-Galactosidase/química , Sistema ABO de Grupos Sanguíneos/metabolismo , Bioensaio , Escherichia coli/enzimologia , Escherichia coli/genética , Fluorescência , Expressão Gênica , Biblioteca Gênica , Glicosídeos/biossíntese , Ensaios de Triagem em Larga Escala , Humanos , Himecromona/metabolismo , Metagenoma , Oligossacarídeos/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa-N-Acetilgalactosaminidase/genética , alfa-N-Acetilgalactosaminidase/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
7.
J Am Chem Soc ; 137(17): 5695-705, 2015 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-25870881

RESUMO

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/metabolismo , Antígenos de Grupos Sanguíneos/química , Configuração de Carboidratos , Sequência de Carboidratos , Eritrócitos/química , Eritrócitos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Polissacarídeos/química , Streptococcus pneumoniae/enzimologia
9.
Anal Biochem ; 444: 67-74, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24121013

RESUMO

Polysialyltransferases (PSTs) assemble polysialic acid (PSA) and have been implicated in many biological processes. For example, certain bacteria such as neuroinvasive Neisseria meningitidis decorate themselves in a PSA capsule to evade the innate immune system. Identifying inhibitors of PSTs therefore represents an attractive therapeutic goal and herein we describe a high-throughput, robust, and sensitive microtiter-plate-based activity assay for PST from N. meningitidis. A trisialyl lactoside (GT3) serving as the acceptor substrate was immobilized on a 384-well plate by click chemistry. Incubation with PST and CMP-sialic acid for 30min resulted in polysialylation. The immobilized PSA was then directly detected using a green fluorescent protein (GFP)-fused PSA-binding protein consisting of the catalytically inactive double mutant of an endosialidase (GFP-EndoNF DM). We report very good agreement between kinetic and inhibition parameters obtained with our on-plate assay versus our in-solution validation assay. In addition we prove our assay is robust and reliable with a Z' score of 0.79. All aspects of our assay are easily scalable owing to optimization trials that allowed immobilization of acceptor substrates prepared from crude reaction mixtures and the use of cell lysates. This assay methodology enables large-scale PST inhibitor screens and can be harnessed for directed evolution screens.


Assuntos
Ensaios Enzimáticos/métodos , Ensaios de Triagem em Larga Escala/métodos , Neisseria meningitidis/enzimologia , Sialiltransferases/metabolismo , Glicosídeos/química , Glicosídeos/isolamento & purificação , Glicosídeos/metabolismo , Estrutura Molecular , Sialiltransferases/antagonistas & inibidores
10.
FEBS J ; 291(15): 3481-3498, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38696354

RESUMO

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators.


Assuntos
Técnicas Biossensoriais , Ácidos Dicarboxílicos , Saccharomyces cerevisiae , Fatores de Transcrição , Técnicas Biossensoriais/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácidos Dicarboxílicos/metabolismo , Ácidos Dicarboxílicos/química , Cristalografia por Raios X , Modelos Moleculares , Ligantes , Conformação Proteica , Ácido Succínico/metabolismo , Ácido Succínico/química , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
11.
Protein Eng Des Sel ; 362023 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-36444941

RESUMO

Many glycosylated small molecule natural products and glycoprotein biologics are important in a broad range of therapeutic and industrial applications. The sugar moieties that decorate these compounds often show a profound impact on their biological functions, thus biocatalytic methods for controlling their glycosylation are valuable. Enzymes from nature are useful tools to tailor bioproduct glycosylation but these sometimes have limitations in their catalytic efficiency, substrate specificity, regiospecificity, stereospecificity, or stability. Enzyme engineering strategies such as directed evolution or semi-rational and rational design have addressed some of the challenges presented by these limitations. In this review, we highlight some of the recent research on engineering enzymes to tailor the glycosylation of small molecule natural products (including alkaloids, terpenoids, polyketides, and peptides), as well as the glycosylation of protein biologics (including hormones, enzyme-replacement therapies, enzyme inhibitors, vaccines, and antibodies).


Assuntos
Produtos Biológicos , Glicosilação , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Especificidade por Substrato , Engenharia de Proteínas , Biocatálise
13.
ACS Chem Biol ; 17(12): 3331-3340, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34751552

RESUMO

Many small molecule natural products are decorated with sugar moieties that are essential for their biological activity. A considerable number of natural product glycosides and their derivatives are clinically important therapeutics. Anthracyclines like daunorubicin and doxorubicin are examples of valuable glycosylated natural products used in medicine as potent anticancer agents. The sugar moiety, l-daunosamine (a highly modified deoxyhexose), plays a key role in the bioactivity of these molecules as evidenced by semisynthetic anthracycline derivatives such as epirubicin, wherein alteration in the configuration of a single stereocenter of the sugar unit generates a chemotherapeutic drug with lower cardiotoxicity. The nucleotide activated sugar donor that provides the l-daunosamine group for attachment to the natural product scaffold in the biosynthesis of these anthracyclines is dTDP-l-daunosamine. In an in vitro system, we have reconstituted the enzymes in the daunorubicin/doxorubicin pathway involved in the biosynthesis of dTDP-l-daunosamine. Through the study of the enzymatic steps in this reconstituted pathway, we have gained several insights into the assembly of this precursor including the identification of a major bottleneck and competing reactions. We carried out kinetic analysis of the aminotransferase that catalyzes a limiting step of the pathway. Our in vitro reconstituted pathway also provided a platform to test the combinatorial enzymatic synthesis of other dTDP-activated deoxyhexoses as potential tools for "glycodiversification" of natural products. To this end, we replaced the stereospecific ketoreductase that acts in the last step of dTDP-l-daunosamine biosynthesis with an enzyme from a heterologous pathway with opposite stereospecificity and found that it is active in the in vitro pathway, demonstrating the potential for the enzymatic synthesis of nucleotide-activated sugars with regio- and stereospecific tailoring.


Assuntos
Produtos Biológicos , Policetídeos , Antraciclinas/metabolismo , Glicosilação , Vias Biossintéticas , Cinética , Daunorrubicina , Antibióticos Antineoplásicos , Doxorrubicina , Carboidratos , Desoxirribonucleotídeos , Nucleotídeos/metabolismo , Açúcares
14.
ACS Synth Biol ; 11(4): 1692-1698, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35316041

RESUMO

Allosteric transcription factor (aTF) biosensors are valuable tools for engineering microbes toward a multitude of applications in metabolic engineering, biotechnology, and synthetic biology. One of the challenges toward constructing functional and diverse biosensors in engineered microbes is the limited toolbox of identified and characterized aTFs. To overcome this, extensive bioprospecting of aTFs from sequencing databases, as well as aTF ligand-specificity engineering are essential in order to realize their full potential as biosensors for novel applications. In this work, using the TetR-family repressor CmeR from Campylobacter jejuni, we construct aTF genetic circuits that function as salicylate biosensors in the model organisms Escherichia coli and Saccharomyces cerevisiae. In addition to salicylate, we demonstrate the responsiveness of CmeR-regulated promoters to multiple aromatic and indole inducers. This relaxed ligand specificity of CmeR makes it a useful tool for detecting molecules in many metabolic engineering applications, as well as a good target for directed evolution to engineer proteins that are able to detect new and diverse chemistries.


Assuntos
Técnicas Biossensoriais , Fatores de Transcrição , Escherichia coli/genética , Escherichia coli/metabolismo , Indóis , Ligantes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Salicilatos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Org Biomol Chem ; 9(7): 2053-6, 2011 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-21340070

RESUMO

Ketoreductase enzymes are responsible for the generation of hydroxyl stereocentres during the biosynthesis of complex polyketide natural products. Previous studies of isolated polyketide ketoreductases have shown that the stereospecificity of ketoreduction can be switched by mutagenesis of selected active site amino acids. We show here that in the context of the intact polyketide synthase multienzyme the same changes do not alter the stereochemical outcome in the same way. These findings point towards additional factors that govern ketoreductase stereospecificity on intact multienzymes in vivo.


Assuntos
Cetonas/química , Policetídeo Sintases/metabolismo , Saccharopolyspora/enzimologia , Domínio Catalítico , Cetonas/metabolismo , Cinética , Estrutura Molecular , Mutação , Oxirredução , Saccharopolyspora/genética , Estereoisomerismo
16.
Molecules ; 16(7): 6092-115, 2011 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-21775938

RESUMO

Polyketides are a diverse class of medically important natural products whose biosynthesis is catalysed by polyketide synthases (PKSs), in a fashion highly analogous to fatty acid biosynthesis. In modular PKSs, the polyketide chain is assembled by the successive condensation of activated carboxylic acid-derived units, where chain extension occurs with the intermediates remaining covalently bound to the enzyme, with the growing polyketide tethered to an acyl carrier domain (ACP). Carboxylated acyl-CoA precursors serve as activated donors that are selected by the acyltransferase domain (AT) providing extender units that are added to the growing chain by condensation catalysed by the ketosynthase domain (KS). The action of ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) activities can result in unreduced, partially reduced, or fully reduced centres within the polyketide chain depending on which of these enzymes are present and active. The PKS-catalysed assembly process generates stereochemical diversity, because carbon-carbon double bonds may have either cis- or trans- geometry, and because of the chirality of centres bearing hydroxyl groups (where they are retained) and branching methyl groups (the latter arising from use of propionate extender units). This review shall cover the studies that have determined the stereochemistry in many of the reactions involved in polyketide biosynthesis by modular PKSs.


Assuntos
Macrolídeos/química , Macrolídeos/metabolismo , Policetídeo Sintases/metabolismo , Aciltransferases/metabolismo , Estrutura Molecular , Estereoisomerismo
17.
Biomicrofluidics ; 13(3): 034106, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31123538

RESUMO

Sialyl-LewisX and LewisX are cell-surface glycans that influence cell-cell adhesion behaviors. These glycans are assembled by α(1,3)-fucosyltransferase enzymes. Their increased expression plays a role in inflammatory disease, viral and microbial infections, and cancer. Efficient screens for specific glycan modifications such as those catalyzed by fucosyltransferases are tended toward costly materials and large instrumentation. We demonstrate for the first time a fucosylation inhibition assay on a digital microfluidic system with the integration of image-based techniques. Specifically, we report a novel lab-on-a-chip approach to perform a fluorescence-based inhibition assay for the fucosylation of a labeled synthetic disaccharide, 4-methylumbelliferyl ß-N-acetyllactosaminide. As a proof-of-concept, guanosine 5'-diphosphate has been used to inhibit Helicobacter pylori α(1,3)-fucosyltransferase. An electrode shape (termed "skewed wave") is designed to minimize electrode density and improve droplet movement compared to conventional square-based electrodes. The device is used to generate a 10 000-fold serial dilution of the inhibitor and to perform fucosylation reactions in aqueous droplets surrounded by an oil shell. Using an image-based method of calculating dilutions, referred to as "pixel count," inhibition curves along with IC50 values are obtained on-device. We propose the combination of integrating image analysis and digital microfluidics is suitable for automating a wide range of enzymatic assays.

18.
ACS Chem Biol ; 14(4): 715-724, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30831024

RESUMO

In cancers, increased fucosylation (attachment of fucose sugar residues) on cell-surface glycans, resulting from the abnormal upregulation of the expression of specific fucosyltransferase enzymes (FUTs), is one of the most important types of glycan modifications associated with malignancy. Fucosylated glycans on cell surfaces are involved in a multitude of cellular interactions and signal regulation in normal biological processes, as well as in disease. For example, sialyl LewisX is a fucosylated cell-surface glycan that is abnormally abundant in some cancers where it has been implicated in facilitating metastasis, allowing circulating tumor cells to bind to the epithelial tissue within blood vessels and invade into secondary sites by taking advantage of glycan-mediated interactions. To identify inhibitors of FUT enzymes as potential cancer therapeutics, we have developed a novel high-throughput assay that makes use of a fluorogenically labeled oligosaccharide as a probe of fucosylation. This probe, which consists of a 4-methylumbelliferyl glycoside, is recognized and hydrolyzed by specific glycoside hydrolase enzymes to release fluorescent 4-methylumbelliferone, yet when the probe is fucosylated prior to treatment with the glycoside hydrolases, hydrolysis does not occur and no fluorescent signal is produced. We have demonstrated that this assay can be used to measure the inhibition of FUT enzymes by small molecules, because blocking fucosylation will allow glycosidase-catalyzed hydrolysis of the labeled oligosaccharide to produce a fluorescent signal. Employing this assay, we have screened a focused library of small molecules for inhibitors of a human FUT enzyme involved in the synthesis of sialyl LewisX and demonstrated that our approach can be used to identify potent FUT inhibitors from compound libraries in microtiter plate format.


Assuntos
Inibidores Enzimáticos/análise , Fucose/química , Fucosiltransferases/antagonistas & inibidores , Fucosiltransferases/metabolismo , Ensaios de Triagem em Larga Escala , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glicosilação , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Triazóis/química , Triazóis/metabolismo
19.
Methods Enzymol ; 597: 25-53, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28935105

RESUMO

Directed evolution is an incredibly powerful strategy for engineering enzyme function. Applying this approach to glycosidases offers enormous potential for the development of highly specialized tools in chemical glycobiology. Performing enzyme directed evolution requires the generation, by random mutagenesis, of mutant libraries from which large numbers of variant enzymes must be screened in high-throughput assays. A structure-guided "semirational" method for library creation allows researchers to target specific amino acid positions for mutagenesis, concentrating mutations where they might be most effective in order to produce mutant libraries of a manageable size, minimizing screening effort while maximizing the chances of finding improved mutants. Well-designed assays, which may use specially prepared substrates, enable efficient screening of these mutant libraries. This chapter will detail general methods in the structure-guided directed evolution of glycosidases, which have previously been employed in engineering a blood group antigen-cleaving enzyme.


Assuntos
Antígenos de Grupos Sanguíneos/química , Evolução Molecular Direcionada , Glicosídeo Hidrolases/química , Engenharia de Proteínas , Antígenos de Grupos Sanguíneos/genética , Biblioteca Gênica , Glicosídeo Hidrolases/genética , Humanos , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica
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