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

Overview

Journal Molecules

Publisher MDPI

Specialty Biology

Date 2018 Jan 11

PMID 29316637

Citations 16

Authors

Piero Leone

Michele Galluccio

Alberto Barbiroli

Ivano Eberini

Maria Tolomeo

Flavia Vrenna

Elisabetta Gianazza

Stefania Iametti

Francesco Bonomi

Cesare Indiveri

Maria Barile

Affiliations

Soon will be listed here.

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 and purified with a yield of 25 mg·L of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (k about 2.8 min), as well as FAD pyrophosphorolysis in a strictly Mg-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.

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