A Regulatory Role of NAD Redox Status on Flavin Cofactor Homeostasis inS. cerevisiaeMitochondria

Teresa Anna Giancaspero,Maria Barile,Vittoria Locato

doi:10.1155/2013/612784

Abstract

Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear in Saccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD+ and NADH according to a noncompetitive inhibition, with Ki values that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae.

Highlights

Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two molecules of pivotal importance for mitochondrial functionality, given their role as redox cofactors of a large number of dehydrogenases, reductases, and oxidases mainly involved in energy production and redox homeostasis [1,2,3,4]
These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae
The differential inhibition by the oxidized and reduced form of NAD, together with the ability of mitochondrial FADppase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allows for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and, possibly, flavoprotein degradation in S. cerevisiae

Summary

Introduction

Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two molecules of pivotal importance for mitochondrial functionality, given their role as redox cofactors of a large number of dehydrogenases, reductases, and oxidases mainly involved in energy production and redox homeostasis [1,2,3,4]. Additional emerging regulatory roles are linked to a number of additional cofactor-dependent events, such as protein folding, apoptosis, gene silencing, transcriptional regulation, DNA repairs and calcium-dependent signaling pathways In many of these processes NAD and FAD are involved in nonredox reactions (for recent reviews see [2, 5, 6]). A diphosphatase (pyrophosphatase), performing an enzymatic activity towards NADH, as preferred substrate, and giving NMNH and AMP as products, has been characterized in yeast as belonging to the NUDIX hydrolase family (EC 3.6.1.-) [27] This enzyme, namely, Npy1p encoded by YGL067W, is able to catalyze NAD+ hydrolysis and very weakly FAD hydrolysis; it was reported to be located in peroxisomes, active at alkaline pH, and strongly inhibited by F−. The differential inhibition by the oxidized and reduced form of NAD, together with the ability of mitochondrial FADppase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allows for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and, possibly, flavoprotein degradation in S. cerevisiae

Materials and Methods

Results and Discussion

Conclusions