Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis.

Teresa A Giancaspero,Roderich Brandsch,Matilde Colella,Graziana Difonzo,Carmen Brizio,Stefania Iametti,Piero Leone,Francesco Bonomi,Giuseppina M Fiorino,Maria Barile

doi:10.3389/fchem.2015.00030

Abstract

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD “chaperone.” The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

Highlights

The crucial role of the water soluble vitamin B2 or riboflavin (Rf) in cell metabolism is linked to Rf conversion into the enzyme cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)
Subcellular Localization of FAD synthase (FADS) in Human Liver Carcinoma (HepG2) Cells As stated in the Introduction, the first challenge of this paper is to provide further evidence of multi-compartmentalisation of FADS in human cells
The presence of FADS in both the mitochondrion and nucleus was demonstrated for the first time in freshly isolated rat liver fractions (Giancaspero et al, 2013)

Summary

Introduction

The crucial role of the water soluble vitamin B2 or riboflavin (Rf) in cell metabolism is linked to Rf conversion into the enzyme cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Subcellular localisation of FAD forming enzymes in mammalian, yeast and plant cells (Barile et al, 1993, 2000; Pallotta et al, 1998; Giancaspero et al, 2009) has been recently addressed at the molecular level (Bafunno et al, 2004; Torchetti et al, 2010; Liuzzi et al, 2012). These studies demonstrated that mitochondria possess their own FADS isoforms. The involvement of additional factors in vivo—including Hsp and Hsp10—is discussed

Materials and Methods

Results and Discussion

Conclusion