Characterization of a non-nudix pyrophosphatase points to interplay between flavin and NAD(H) homeostasis in Saccharomyces cerevisiae

Abstract

The flavin cofactors FMN and FAD are required for a wide variety of biological processes, however, little is known about their metabolism. Here, we report the cloning and biochemical characterization of the Saccharomyces cerevisiae pyrophosphatase Fpy1p. Genetic and functional studies suggest that Fpy1p may play a key role in flavin metabolism and is the first-reported non-Nudix superfamily enzyme to display FAD pyrophosphatase activity. Characterization of mutant yeast strains found that deletion of fpy1 counteracts the adverse effects that are caused by deletion of flx1, a known mitochondrial FAD transporter. We show that Fpy1p is capable of hydrolyzing FAD, NAD(H), and ADP-ribose. The enzymatic activity of Fpy1p is dependent upon the presence of K+ and divalent metal cations, with similar kinetic parameters to those that have been reported for Nudix FAD pyrophosphatases. In addition, we report that the deletion of fpy1 intensifies the FMN-dependence of null mutants of the riboflavin kinase Fmn1p, demonstrate that fpy1 mutation abolishes the decreased fitness resulting from the deletion of the flx1 ORF, and offer a possible mechanism for the genetic interplay between fpy1, flx1 and fmn1.

A higher plant FAD synthetase is fused to an inactivated FAD pyrophosphatase

“…S1 ). As previously reported, human FAD synthetases, hFADS1 and hFADS2, transcript variants encoded by a single gene, are also homologous to S. cerevisiae FAD1, and both variants include an extra domain with homology to FPY1, as do homologs in other animal species ( 36 ). These contrasting architectures across multiple kingdoms of life are indicative of convergent evolution toward fusion of the two proteins.…”

“…The activity of this essential enzyme ( 35 ) is dependent on Mg 2+ ( 26 ). Humans and other animals possess a sequence homolog with a substantial, up to 298 residue, extra domain on the N terminus ( 36 ), which was recently identified as an FAD pyrophosphatase ( 37 , 38 ). The human protein is present as at least two transcript variants, both of which have been shown to also encode Mg 2+ -dependent enzymes, localized in mitochondria and cytosol, respectively ( 23 , 24 , 25 , 39 ).…”

Development of Novel Experimental Models to Study Flavoproteome Alterations in Human Neuromuscular Diseases: The Effect of Rf Therapy

“…Isoform 2 has been over-produced and purified in its catalytically active form: it contains redox-sensitive cysteines which make hFADS2 a putative redox-sensor [76]. In [35], the novel feature of hFADS2 to catalyze FAD hydrolysis has been characterized especially for its ability to create a link between the flavin and the NAD world as already proposed in yeast [7,77] Besides regulating FAD production/hydrolysis, FADS also takes part in cofactor delivery to the appropriate apo-flavoenzymes during holoenzyme biogenesis, operating in a flavinylation machinery as a FAD "chaperone" [78,79].…”

“…Isoform 2 has been over-produced and purified in its catalytically active form: it contains redox-sensitive cysteines which make hFADS2 a putative redox-sensor [ 76 ]. In [ 35 ], the novel feature of hFADS2 to catalyze FAD hydrolysis has been characterized especially for its ability to create a link between the flavin and the NAD world as already proposed in yeast [ 7 , 77 ]…”

“…The mammalian FADHy domain has, indeed, a single domain enzymatic counterpart in yeast, namely the product of FPY1, which performs a non-Nudix diphosphatase activity [ 77 ] capable of hydrolysing in vitro FAD, NAD(H), and ADP-ribose in presence of K + and divalent cations. Indeed, our group observed that S. cerevisiae mitochondria (SCM) are able to perform FAD hydrolysis via an enzymatic activity which is different from the already characterized Nudix hydrolases; since this activity is regulated by the NAD redox status, an experimental link between NAD and FAD mitochondrial world has been proposed [ 174 ].…”