Data/hora: 07/06/2024 02:37:58

Provedor de dados: | 5
País: | France
Título: | Combined Whole-Cell High-Throughput Functional Screening for Identification of New Nicotinamidases/Pyrazinamidases in Metagenomic/Polygenomic Libraries
Autores: | Zapata-perez, Ruben; Garcia-saura, Antonio G.; Jebbar, Mohamed; Golyshin, Peter N.; Sanchez-ferrer, Alvaro.
Data: | 2016-11
Ano: | 2016
Palavras-chave: | Amidohydrolases; Nicotinamidase; Functional screening; Metagenome; Library; High-throughput screening assays.
Resumo: | Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide (NAM) to produce ammonia and nicotinic acid (NA). These enzymes are an essential component of the NAD(+) salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or NA) to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside (SNP). After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the SNP method allowed the finding of the first nicotinamidase with balanced catalytic efficiency toward NAM (nicotinamidase activity) and pyrazinamide (pyrazinamidase activity). Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.
Tipo: | Text
Idioma: | Inglês

Identificador:

http://archimer.ifremer.fr/doc/00362/47269/47232.pdf

http://archimer.ifremer.fr/doc/00362/47269/47233.pdf

DOI:10.3389/fmicb.2016.01915

http://archimer.ifremer.fr/doc/00362/47269/

Editor: | Frontiers Media Sa

Relação:

info:eu-repo/grantAgreement/EC/FP7/226977/EU//MAMBA

info:eu-repo/grantAgreement/EC/FP7/634486/EU//INMARE

Formato: | application/pdf
Fonte: | Frontiers In Microbiology (1664-302X) (Frontiers Media Sa), 2016-11 , Vol. 7 , N. 1915 , P. 1-13

Fonte:

2016 Zapata-Pérez, García-Saura, Jebbar, Golyshin and Sánchez-Ferrer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not...

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