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Volume 23
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10.3390/molecules23010116
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Article

Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

by
Piero Leone
1,†,
Michele Galluccio
2,†,
Alberto Barbiroli
3,
Ivano Eberini
4,
Maria Tolomeo
1,
Flavia Vrenna
2,
Elisabetta Gianazza
4,
Stefania Iametti
3,
Francesco Bonomi
3,
Cesare Indiveri
2,5 and
Maria Barile
1,*
1
Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, via Orabona, 4, I-70126 Bari, Italy
2
Department of Biology, Ecology and Earth Science (DiBEST), Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4c, I-87036 Arcavacata di Rende, Italy
3
Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente (DeFENS), Università degli Studi di Milano, via G. Celoria 2, I-20133 Milano, Italy
4
Gruppo di Studio per la Proteomica e la Struttura di Proteine, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Università degli Studi di Milano, via Balzaretti 9, I-20133 Milano, Italy
5
Institute of Biomembranes, Bioenergetics and Molecular Biotechnology (IBIOM)—CNR, Via Giovanni Amendola 165/A-70126 Bari, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2018, 23(1), 116; https://doi.org/10.3390/molecules23010116
Received: 2 December 2017 / Revised: 30 December 2017 / Accepted: 4 January 2018 / Published: 6 January 2018
(This article belongs to the Special Issue Flavoenzymes)
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Abstract

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3′-phosphoadenosine 5′-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L−1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (kcat about 2.8 min−1), as well as FAD pyrophosphorolysis in a strictly Mg2+-dependent manner. The synthesis of FAD is inhibited by HgCl2. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed. View Full-Text
Keywords: flavin; riboflavin; flavoprotein; FAD synthase; protein modeling; over-expression; E. coli flavin; riboflavin; flavoprotein; FAD synthase; protein modeling; over-expression; E. coli
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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MDPI and ACS Style

Leone, P.; Galluccio, M.; Barbiroli, A.; Eberini, I.; Tolomeo, M.; Vrenna, F.; Gianazza, E.; Iametti, S.; Bonomi, F.; Indiveri, C.; Barile, M. Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein? Molecules 2018, 23, 116. https://doi.org/10.3390/molecules23010116

AMA Style

Leone P, Galluccio M, Barbiroli A, Eberini I, Tolomeo M, Vrenna F, Gianazza E, Iametti S, Bonomi F, Indiveri C, Barile M. Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein? Molecules. 2018; 23(1):116. https://doi.org/10.3390/molecules23010116

Chicago/Turabian Style

Leone, Piero, Michele Galluccio, Alberto Barbiroli, Ivano Eberini, Maria Tolomeo, Flavia Vrenna, Elisabetta Gianazza, Stefania Iametti, Francesco Bonomi, Cesare Indiveri, and Maria Barile. 2018. "Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?" Molecules 23, no. 1: 116. https://doi.org/10.3390/molecules23010116

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