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. 2018 Jun 14;13(6):e0198787.
doi: 10.1371/journal.pone.0198787. eCollection 2018.

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

Joseph H Lynch  1 , Na Sa  1 , Sompop Saeheng  1 , Nadia Raffaelli  2 , Sanja Roje  1
Affiliations

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

Joseph H Lynch et al. PLoS One. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Analysis of substrate binding of Fpy1p.
(A) Substrate specificity of Fpy1p. Data are expressed as a percent of activity relative to that observed when FAD is used as substrate with each metal ion, and is given as the average ± S.E. of three triplicate determinations in which AMP production was quantified. Assays were performed with 100 μM substrate along with either 4 mM CoCl2 (cobalt) or 10 mM MgCl2 (magnesium). (B) FAD pyrophosphatase activity of Fpy1p in the presence of potential inhibitors. Data are shown as the average ± S.E. of three triplicate determinations in which FMN production was quantified. Assays were performed using 4 mM CoCl2, 20 μM FAD, and 50 μM lumiflavin or nicotinamide when specified. Higher concentrations were not used due to limited solubility of lumiflavin.
Fig 2
Fig 2. FAD pyrophosphatase activity of Fpy1p as a function of metal ion concentration.
Data are shown as the average ± S.E. of three triplicate determinations. Assays were performed using either CoCl2 (solid circles) or MgCl2 (open circles) in the presence of 50 μM FAD.
Fig 3
Fig 3. Potassium requirement for the FAD pyrophosphatase activity of Fpy1p.
(A) Enzyme activity in the absence or presence of 200 mM KCl and/or 200 mM NaCl when assayed with 4 mM CoCl2. (B) Enzyme activity in the absence or presence of 200 mM KCl and/or 200 mM NaCl when assayed with 8 mM MgCl2. (C) Enzyme activity as a function of KCl concentration in the presence of 4 mM CoCl2 (solid circles) or 10 mM MgCl2 (open circles). Data are given as the average ± S.E. of three triplicate determinations.
Fig 4
Fig 4. Effect of pH on FAD pyrophosphatase activity of Fpy1p.
The enzymatic activity of Fpy1p was determined as described under Experimental Procedures, with the exception that MHC-KOH buffer (100 mM MES, 100 mM Hepes, 100 mM CHES) was used in place of HEPES-KOH. Assays included 50 μM FAD and either 4 mM CoCl2 (solid circles) or 10 mM MgCl2 (open circles). Data are given as the average ± S.E. of three triplicate determinations.
Fig 5
Fig 5. Growth analysis of wild type and mutant yeast under different conditions.
(A) Colony size for wild type yeast and the Δfpy1, Δfmn1, and Δfmn1/Δfpy1 mutants grown on YPD supplemented with 4 mM FMN for 48 hours. (B) Wild type yeast and the Δfpy1, Δflx1, and Δflx1/Δfpy1 mutants grown on either SD/MSG medium for 72 hours or (C) YPD medium for 48 hours. Colony size was measured as described in Experimental Procedures. Data are given as the average ± S.E of 6–18 colonies per mutant line per condition tested. Different letters within each chart signify data which are statistically different (p < .05) based on one-way ANOVA with Holm-Šidák pairwise comparison.
Fig 6
Fig 6. Growth of wild-type and mutant yeast under different conditions.
The OD600 values were measured in a microplate (path length ~0.6 cm). Yeast were grown by flask culture in YP medium (1% yeast extract, 2% peptone) supplemented with 2% glucose (A, B), 3% glycerol (C); and 1% sodium acetate (D), under either normal (A, C, D) or hypoxic (B) conditions. Data are given as the average ± S.E. of three separate cultures. Symbols are: solid circles, wild-type; open circles, Δfpy1; solid triangles, Δflx1; open triangles, Δflx1/Δfpy1.
Fig 7
Fig 7. Comparison of exponential growth rates of yeast grown on YP + 1% sodium acetate.
The portion of the growth curves which appeared linear on a semi-log plot (6–20 hours for wild type and Δfpy1, 36–52 hours for Δflx1 and Δflx1/Δfpy1) were fit by least squares regression to the equation for exponential growth. Data for each condition are given as the average ± SE of three individual cultures. Different letters signify data which are statistically different (p < .05) based on one-way ANOVA with Holm-Šidák pairwise comparison.
Fig 8
Fig 8. Known routes for production of NADH and subsequent contribution to mitochondrial electron transport chain from catabolism of various carbon sources.
Included are glycolysis (blue), the glyoxylate cycle (rose), fermentation (green), and the citric acid cycle (orange), as well as the connecting steps between the pathways. Also shown are the routes for entry of galactose and glycerol catabolic products into the core glycolysis pathway. Abbreviations not described in the text are as follows: Fru-6-P, fructose-6-phosphate; Fru-1,6-BP, fructose-1,6-bisphophate; GAP, glyceraldehyde-3-phosphate; 1,3-BPG, 1,3-bisphosphoglycerate; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Gal-6-P, galactose-6-phosphate; Glu-1-P, glucose-1-phosphate; UDP-Glu, UDP-glucose; UDP-Gal, UDP-galactose; Acetyl-Carn, acetylcarnitine; α-KG, alpha-ketoglutarate; Succ-CoA, succinyl-CoA; GUT2, mitochondrial G3P dehydrogrenase; ETC, electron transport chain.
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This work was supported by the United States National Institute of Food and Agriculture (https://nifa.usda.gov/) under National Research Initiative Competitive Grant 2007-35318-18438, the National Science Foundation (https://www.nsf.gov/) Grant MCB-1052492, and the Italian Ministry of Foreign Affairs (http://www.esteri.it/mae/en) “Direzione Generale per la Promozione del Sistema Paese”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.