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A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.

ABSTRACT: Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNF?-derived peptide. This substrate is well recognized by human sirtuins 1-6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000?M(-1)s(-1). These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600?nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNF? (80?nM).

SUBMITTER: Schuster S

PROVIDER: S-EPMC4778124 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.

Schuster Sabine S Roessler Claudia C Meleshin Marat M Zimmermann Philipp P Simic Zeljko Z Kambach Christian C Schiene-Fischer Cordelia C Steegborn Clemens C Hottiger Michael O MO Schutkowski Mike M

Scientific reports 20160304

Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescenc ...[more]

PMID: 26940860

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Project description:UnlabelledQuorum sensing, the bacterial cell-cell communication by small molecules, controls important processes such as infection and biofilm formation. Therefore, it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivating N-acyl-l-homoserine lactones, the most common class of communication molecules among Gram-negative proteobacteria, mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However, identification, characterization, and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weaknesses. Here, a (bio)chemical activity assay is described, which perfectly complements the other methods in this field. It enables continuous and high-throughput activity measurements of purified and unpurified quorum-quenching enzymes within several minutes. For this, the reaction products released by quorum-quenching lactonases and quorum-quenching acylases are converted either by a secondary enzyme or by autohydrolysis to l-homoserine. In turn, l-homoserine is detected by the previously described calcein assay, which is sensitive to α-amino acids with free N and C termini. Besides its establishment, the method was applied to the characterization of three previously undescribed quorum-quenching lactonases and variants thereof and to the identification of quorum-quenching acylase-expressing Escherichia coli clones in an artificial library. Furthermore, this study indicates that porcine aminoacylase 1 is not active toward N-acyl-l-homoserine lactones as published previously but instead converts the autohydrolysis product N-acyl-l-homoserine.ImportanceIn this study, a novel method is presented for the identification, characterization, and optimization of quorum-quenching enzymes that are active toward N-acyl-l-homoserine lactones. These are the most common communication molecules among Gram-negative proteobacteria. The activity assay is a highly valuable complement to the available analytical tools in this field. It will facilitate studies on the environmental impact of quorum-quenching enzymes and contribute to the development of therapeutic and technical applications of this promising enzyme class.

Dataset Information

A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.

Publications

A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.

Similar Datasets

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