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1.
Methods Enzymol ; 705: 347-376, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39389669

RESUMO

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1, APEX1, REF1, HAP1) is an abasic site-specific endonuclease holding critical roles in numerous biological functions including base excision repair, the DNA damage response, redox regulation of transcription factors, RNA processing, and gene regulation. Pathologically, APE1 expression and function is linked with numerous human diseases including cancer, highlighting the importance of sensitive and quantitative assays to measure APE1 activity. Here, we summarize biochemical and biological roles for APE1 and expand on the discovery of APE1 inhibitors. Finally, we highlight the development of assays to monitor APE1 activity, detailing a recently improved and stabilized DNA Repair Molecular Beacon assay to analyze APE1 activity. The assay is amenable to analysis of purified protein, to measure changes in APE1 activity in cell lysates, to monitor human patient samples for defects in APE1 function, or the cellular and biochemical response to APE1 inhibitors.


Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Ensaios Enzimáticos , Inibidores Enzimáticos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , Humanos , Inibidores Enzimáticos/farmacologia , Ensaios Enzimáticos/métodos , Dano ao DNA
2.
Methods Enzymol ; 705: 311-345, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39389668

RESUMO

In recent years, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become ever more apparent. This growing awareness and lack of inhibitory drugs has created a distinct need for biochemical tools that can be used to identify and characterize potential inhibitors of this family of enzymes. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. The RADD assay provides a rapid, real-time fluorescence readout of APOBEC3 DNA deamination and serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit. This method improves upon contemporary DNA deamination assays by offering a more rapid and quantifiable readout as well as providing a platform that is readily adaptable to a high-throughput format for inhibitor discovery. In this chapter we provide a detailed guide for the usage of the RADD assay for the characterization of APOBEC3 enzymes and potential inhibitors.


Assuntos
DNA , Transferência Ressonante de Energia de Fluorescência , Humanos , Transferência Ressonante de Energia de Fluorescência/métodos , DNA/metabolismo , Desaminação , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Desaminases APOBEC/metabolismo
3.
Int J Mol Sci ; 25(17)2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39273463

RESUMO

Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones.


Assuntos
Fosfatidilcolinas , Fosfolipases A2 , Fosfolipases A2/metabolismo , Fosfolipases A2/química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Cinética , Micelas , Lipossomos/química , Concentração de Íons de Hidrogênio , Ensaios Enzimáticos/métodos , Octoxinol/química
4.
Methods Enzymol ; 703: 147-166, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39260994

RESUMO

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.


Assuntos
Espectrometria de Massas , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Massas/métodos , Cobalto/química , Cobalto/metabolismo , Dioxigenases/metabolismo , Dioxigenases/química , Ensaios Enzimáticos/métodos , Oxigênio/metabolismo , Oxirredução , Espectrometria de Massa com Cromatografia Líquida
5.
Methods Enzymol ; 703: 87-120, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39261005

RESUMO

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.


Assuntos
5-Metilcitosina , Proteínas de Ligação a DNA , Dioxigenases , Proteínas Proto-Oncogênicas , Dioxigenases/metabolismo , Dioxigenases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análise , 5-Metilcitosina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ensaios Enzimáticos/métodos , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Metilação de DNA , Citosina/análogos & derivados , Citosina/análise , Citosina/metabolismo , Citosina/química , Oxirredução
6.
Methods Enzymol ; 703: 167-192, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39260995

RESUMO

Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.


Assuntos
Oxigenases , Pseudomonas fluorescens , Pseudomonas fluorescens/enzimologia , Oxigenases/metabolismo , Oxigenases/química , Dioxigenases/metabolismo , Dioxigenases/química , Dioxigenases/genética , Ensaios Enzimáticos/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de Elétrons
7.
Methods Enzymol ; 704: 173-198, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39300647

RESUMO

α-Ketoglutarate-dependent non-heme iron (α-KG NHI) oxygenases compose one of the largest superfamilies of tailoring enzymes that play key roles in structural and functional diversifications. During the biosynthesis of meroterpenoids, α-KG NHI oxygenases catalyze diverse types of chemical reactions, including hydroxylation, desaturation, epoxidation, endoperoxidation, ring-cleavage, and skeletal rearrangements. Due to their catalytic versatility, keen attention has been focused on functional analyses of α-KG NHI oxygenases. This chapter provides detailed methodologies for the functional analysis of the fungal α-KG NHI oxygenase SptF, which plays an important role in the structural diversification of andiconin-derived meroterpenoids. The procedures included describe how to prepare the meroterpenoid substrate using a heterologous fungal host, measure the in vitro enzymatic activity of SptF, and how to perform structural and mutagenesis studies on SptF. These protocols are also applicable to functional analyses of other α-KG NHI oxygenases.


Assuntos
Ácidos Cetoglutáricos , Terpenos , Terpenos/metabolismo , Terpenos/química , Ácidos Cetoglutáricos/metabolismo , Oxigenases/metabolismo , Oxigenases/genética , Oxigenases/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Ferroproteínas não Heme/metabolismo , Ferroproteínas não Heme/química , Ferroproteínas não Heme/genética , Fungos/metabolismo , Fungos/genética , Fungos/enzimologia , Ensaios Enzimáticos/métodos , Especificidade por Substrato
8.
Methods Enzymol ; 704: 199-232, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39300648

RESUMO

Iron and 2-oxoglutarate dependent (Fe/2OG) enzymes exhibit an exceedingly broad reaction repertoire. The most prevalent reactivity is hydroxylation, but many other reactivities have also been discovered in recent years, including halogenation, desaturation, epoxidation, endoperoxidation, epimerization, and cyclization. To fully explore the reaction mechanisms that support such a diverse reactivities in Fe/2OG enzyme, it is necessary to utilize a multi-faceted research methodology, consisting of molecular probe design and synthesis, in vitro enzyme assay development, enzyme kinetics, spectroscopy, protein crystallography, and theoretical calculations. By using such a multi-faceted research approach, we have explored reaction mechanisms of desaturation and epoxidation catalyzed by a bi-functional Fe/2OG enzyme, AsqJ. Herein, we describe the experimental protocols and computational workflows used in our studies.


Assuntos
Ferro , Ácidos Cetoglutáricos , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Ferro/química , Ferro/metabolismo , Cinética , Cristalografia por Raios X/métodos , Ensaios Enzimáticos/métodos , Hidroxilação , Modelos Moleculares
9.
Methods Enzymol ; 704: 313-344, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39300654

RESUMO

Aspartate/asparagine-ß-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer. This chapter describes methods for the production and purification of soluble constructs of recombinant human AspH suitable for biochemical and crystallographic studies. The chapter also describes efficient methods for performing turnover and inhibition assays which monitor catalysis of isolated recombinant human AspH in vitro using solid phase extraction coupled to mass spectrometry (SPE-MS). The SPE-MS assays employ synthetic disulfide- or thioether-bridged macrocyclic oligopeptides as substrates; a macrocycle is an apparently essential requirement for productive AspH catalysis and mimics an EGFD disulfide isomer that is not typically observed in crystal and NMR structures. SPE-MS assays can be used to monitor catalysis of 2OG oxygenases other than AspH; the methods described herein are representative for 2OG oxygenase SPE-MS assays useful for performing kinetic and/or inhibition studies.


Assuntos
Oxigenases de Função Mista , Proteínas Recombinantes , Humanos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Ensaios Enzimáticos/métodos , Extração em Fase Sólida/métodos , Espectrometria de Massas/métodos , Catálise , Cinética , Asparagina/metabolismo , Asparagina/química , Hidroxilação , Especificidade por Substrato , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Membrana , Proteínas Musculares
10.
Methods Enzymol ; 703: 51-63, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39261003

RESUMO

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection. This method can be performed in anaerobic glove bag settings. It requires readily available HPLC instrumentation for ligand quantitation and is adaptable to meet the needs of a variety of substrate affinity measurements.


Assuntos
Diálise , Cromatografia Líquida de Alta Pressão/métodos , Especificidade por Substrato , Diálise/métodos , Ligação Proteica , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/instrumentação , Cinética , Lisina/metabolismo , Lisina/química , Oxirredutases/metabolismo , Oxirredutases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ferro/metabolismo , Ferro/química
11.
Methods Enzymol ; 704: 291-312, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39300652

RESUMO

The article reports methods for the expression and assay of 9-cis-epoxycarotenoid cleavage dioxygenase (NCED), an enzyme involved in the biosynthesis of phytohormone abscisic acid in plants. A method for the preparation of the unstable substrate 9'-cis-neoxanthin from fresh spinach is described. The inhibition of Solanum lycopersicum NCED by a series of aryl hydroxamic acid inhibitors is illustrated, and inhibitors D2 and D4 are assayed against NCED isozymes from Zea mays.


Assuntos
Dioxigenases , Solanum lycopersicum , Zea mays , Dioxigenases/metabolismo , Dioxigenases/antagonistas & inibidores , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/enzimologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Ensaios Enzimáticos/métodos , Proteínas de Plantas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/química
12.
Methods Enzymol ; 704: 91-111, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39300658

RESUMO

Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.


Assuntos
Aziridinas , Ciclopropanos , Ácidos Cetoglutáricos , Ciclopropanos/química , Ciclopropanos/metabolismo , Aziridinas/química , Aziridinas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Ferro/química , Ferro/metabolismo , Ferroproteínas não Heme/química , Ferroproteínas não Heme/metabolismo , Biocatálise , Ensaios Enzimáticos/métodos , Catálise
13.
Methods Enzymol ; 704: 113-142, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39300645

RESUMO

Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.


Assuntos
Proteínas de Bactérias , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Streptomyces/genética , Streptomyces/enzimologia , Streptomyces/metabolismo , Oxigenases/metabolismo , Oxigenases/genética , Oxigenases/química , Oxigenases/isolamento & purificação , Ensaios Enzimáticos/métodos , Oxazinas/química , Oxazinas/metabolismo , Ferro/metabolismo , Ferro/química , Escherichia coli/genética , Escherichia coli/metabolismo , Ferroproteínas não Heme/metabolismo , Ferroproteínas não Heme/química , Ferroproteínas não Heme/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/química
14.
Methods Enzymol ; 704: 39-58, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39300656

RESUMO

Non-heme iron oxygenases constitute a versatile enzyme family that is crucial for incorporating molecular oxygen into diverse biomolecules. Despite their importance, only a limited number of these enzymes have been structurally and functionally characterized. Surprisingly, there remains a significant gap in understanding how these enzymes utilize a typical architecture and reaction mechanism to catalyze a wide range of reactions. Improving our understanding of these catalysts holds promise for advancing both fundamental enzymology and practical applications. This chapter aims to outline methods for heterologous expression, enzyme preparation, in vitro enzyme assays, and crystallization of biphenyl dioxygenase, phthalate dioxygenase and terephthalate dioxygenase. These enzymes catalyze the dihydroxylation of biphenyl, phthalate and terephthalate molecules, serving as a model for functional and structural analysis of other non-heme iron oxygenases.


Assuntos
Compostos de Bifenilo , Cristalização , Ácidos Ftálicos , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Compostos de Bifenilo/química , Cinética , Cristalização/métodos , Dioxigenases/química , Dioxigenases/metabolismo , Dioxigenases/genética , Ferro/química , Ferro/metabolismo , Cristalografia por Raios X/métodos , Ensaios Enzimáticos/métodos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Oxigenases
15.
Methods Enzymol ; 704: 345-361, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39300655

RESUMO

The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase utilize a non-heme iron to catalyze the hydroxylation of the aromatic rings of their amino acid substrates, with a tetrahydropterin serving as the source of the electrons necessary for the monooxygenation reaction. These enzymes have been subjected to a variety of biochemical and biophysical approaches, resulting in a detailed understanding of their structures and mechanism. We summarize here the experimental approaches that have led to this understanding.


Assuntos
Fenilalanina Hidroxilase , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/metabolismo , Fenilalanina Hidroxilase/genética , Humanos , Triptofano Hidroxilase/metabolismo , Triptofano Hidroxilase/química , Tirosina 3-Mono-Oxigenase/metabolismo , Tirosina 3-Mono-Oxigenase/química , Animais , Ensaios Enzimáticos/métodos
16.
Methods Mol Biol ; 2851: 115-123, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39210176

RESUMO

A notable characteristic of amino acids is their optical isomerism, existing as L-form and D-form. Proteins are composed exclusively of L-form amino acids. However, recently, it is reported that D-alanine is evaluated particularly highly in terms of sensory evaluation. D-body amino acids convert L-body amino acid proteolysis from a substrate such as foods during fermentation of lactic acid bacteria. This chapter presents a description of methods used for D-alanine racemase assays in the solution producing by lactic acid bacteria (LAB) using D-amino acid oxidase and lactic acid dehydrogenase via a NADH oxidoreduction system.


Assuntos
Alanina Racemase , NAD , Oxirredução , NAD/metabolismo , Alanina Racemase/metabolismo , Alanina Racemase/genética , Lactobacillales/metabolismo , Lactobacillales/enzimologia , Ensaios Enzimáticos/métodos , D-Aminoácido Oxidase/metabolismo , L-Lactato Desidrogenase/metabolismo
17.
Methods Enzymol ; 702: 247-280, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39155115

RESUMO

Siderophores are essential molecules released by some bacteria and fungi in iron-limiting environments to sequester ferric iron, satisfying metabolic needs. Flavin-dependent N-hydroxylating monooxygenases (NMOs) catalyze the hydroxylation of nitrogen atoms to generate important siderophore functional groups such as hydroxamates. It has been demonstrated that the function of NMOs is essential for virulence, implicating these enzymes as potential drug targets. This chapter aims to serve as a resource for the characterization of NMO's enzymatic activities using several biochemical techniques. We describe assays that allow for the determination of steady-state kinetic parameters, detection of hydroxylated amine products, measurement of the rate-limiting step(s), and the application toward drug discovery efforts. While not exhaustive, this chapter will provide a foundation for the characterization of enzymes involved in siderophore biosynthesis, allowing for gaps in knowledge within the field to be addressed.


Assuntos
Oxigenases de Função Mista , Sideróforos , Sideróforos/metabolismo , Sideróforos/biossíntese , Oxigenases de Função Mista/metabolismo , Cinética , Hidroxilação , Ensaios Enzimáticos/métodos , Flavinas/metabolismo , Proteínas de Bactérias/metabolismo
18.
Talanta ; 279: 126626, 2024 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-39116732

RESUMO

Thymidine Kinase 1 (TK1) is a pivotal enzyme in fundamental biochemistry and molecular diagnosis, but recognition and molecule detection is a challenging task. Here, we constructed a DNA-integrated hybrid nanochannel sensor for TK1 activity and inhibition assay. Single-stranded DNA containing thymidine was used as a substrate to functionalize the nanochannels, restricting the ion current through channels. With kinase, the thymidine at the termini of the substrate DNA is phosphorylated, elevating surface charge density and mitigating the pore-obstruction effect by increasing transmembrane ion current. The kinase-induced distinctness can be accurately monitored by this hybrid nanodevice, which benefits from its high sensitivity to the change of surface charge. The excellent analytical performance in both kinase enzyme activity and inhibition analysis resulted in efficient and selective evaluation in human serum. Furthermore, compared to current approaches, it greatly simplifies and offers a direct method of analysis, making it a promising sensor technology for cancer management as well as the activities of multiple types of nucleic acid kinases.


Assuntos
Técnicas Biossensoriais , Timidina Quinase , Timidina Quinase/metabolismo , Timidina Quinase/sangue , Técnicas Biossensoriais/métodos , Humanos , Nanoestruturas/química , Ensaios Enzimáticos/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Timidina/química , Limite de Detecção
19.
Methods Mol Biol ; 2851: 87-95, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39210173

RESUMO

Tannin, which is an astringent taste in the mouth, is a polyphenol compound contained in some plants. Tannin causes denaturation of proteins of the tongue or oral mucosa. Tannase, a hydrolase that cleaves carboxylic ester bonds specifically, is used in many industrial fields. Some tannase (tannin acyl hydrolase, EC3.1.1.20) is used widely to prevent or reduce creaming of some foods and beverages. Because some tannins are formed of insoluble salts combined with protein, they reduce creaming such as the white hazing of iced tea. Moreover, they can clarify beverages such as fruit juices during wine and beer production. Tannase is produced by microorganisms under conditions with tannic acid present, mainly from plants. Tannase characteristics differ according to its microorganism of origin. Therefore, it is important to study the microbes used as lactic acid bacteria (LAB), evaluate new methods of tannase assay, and apply them in food or other industries. In this chapter, assay of tannase in LAB is demonstrated using methyl gallate as substrate, with color development by rhodanine and potassium hydroxide solution, using a spectrophotometer. Actual data of high tannase-producing LAB, Lactobacillus plantarum, and enzyme characteristics in optimum conditions are presented in this chapter.


Assuntos
Hidrolases de Éster Carboxílico , Lactobacillus plantarum , Hidrolases de Éster Carboxílico/metabolismo , Lactobacillus plantarum/enzimologia , Lactobacillus plantarum/metabolismo , Ensaios Enzimáticos/métodos , Taninos/metabolismo , Taninos/química
20.
Methods Enzymol ; 702: 1-19, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39155107

RESUMO

The biosynthesis of many bacterial siderophores employs a member of a family of ligases that have been defined as NRPS-independent siderophore (NIS) synthetases. These NIS synthetases use a molecule of ATP to produce an amide linkage between a carboxylate and an amine. Commonly used carboxylate substrates include citrate or α-ketoglutarate, or derivatives thereof, while the amines are often hydroxamate derivatives of lysine or ornithine, or their decarboxylated forms cadaverine and putrescine. Enzymes that employ three substrates to catalyze a reaction may proceed through alternate mechanisms. Some enzymes use sequential mechanisms in which all three substrates bind prior to any chemical steps. In such mechanisms, substrates can bind in a random, ordered, or mixed fashion. Alternately, other enzymes employ a ping-pong mechanism in which a chemical step occurs prior to the binding of all three substrates. Here we describe an enzyme assay that will distinguish among these different mechanisms for the NIS synthetase, using IucA, an enzyme involved in the production of aerobactin, as the model system.


Assuntos
Peptídeo Sintases , Sideróforos , Sideróforos/metabolismo , Sideróforos/química , Peptídeo Sintases/metabolismo , Peptídeo Sintases/química , Cinética , Especificidade por Substrato , Ensaios Enzimáticos/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ácidos Cetoglutáricos/metabolismo , Ligases/metabolismo , Ligases/química
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