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

The riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A. thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation. Like human FAD synthetase, AtFADS1 exists as an apparent fusion with an ancestral FAD pyrophosphatase, a feature that is conserved across plants. However, we detected no pyrophosphatase activity with AtFADS1, consistent with an observed loss of a key catalytic residue in higher plant evolutionary history. In contrast, we determined that algal FADS1 retains both FAD synthetase and pyrophosphatase activity. We discuss the implications, including the potential for yet-unstudied biologically relevant noncatalytic functions, and possible evolutionary pressures that have led to the loss of FAD pyrophosphatase activity, yet universal retention of an apparently nonfunctional domain in FADS of land plants. The riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A. thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation. Like human FAD synthetase, AtFADS1 exists as an apparent fusion with an ancestral FAD pyrophosphatase, a feature that is conserved across plants. However, we detected no pyrophosphatase activity with AtFADS1, consistent with an observed loss of a key catalytic residue in higher plant evolutionary history. In contrast, we determined that algal FADS1 retains both FAD synthetase and pyrophosphatase activity. We discuss the implications, including the potential for yet-unstudied biologically relevant noncatalytic functions, and possible evolutionary pressures that have led to the loss of FAD pyrophosphatase activity, yet universal retention of an apparently nonfunctional domain in FADS of land plants. Flavin adenine dinucleotide (FAD) and FMN are vital to all living organisms because of their function as irreplaceable cofactors for enzymes participating in a wide variety of metabolic processes. These processes include, but are not limited to, mitochondrial electron transport, photosynthesis, fatty acid oxidation, protein folding, chromatin remodeling, and metabolism of several other biologically relevant compounds, such as nucleotides, amino acids, antioxidants, and other cofactors (1Massey V. Introduction: flavoprotein structure and mechanism.FASEB J. 1995; 9: 473-475Crossref PubMed Scopus (97) Google Scholar, 2Eggers R. Jammer A. Jha S. Kerschbaumer B. Lahham M. 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FMN and FAD are derived from riboflavin, for which the de novo synthesis pathway in bacteria, yeast, and plants has been characterized and extensively reviewed (5Roje S. Vitamin B biosynthesis in plants.Phytochemistry. 2007; 68: 1904-1921Crossref PubMed Scopus (139) Google Scholar, 9Sa N. Rawat R. Thornburg C. Walker K.D. Roje S. Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana.Plant J. 2016; 88: 705-716Crossref PubMed Scopus (26) Google Scholar, 10Bacher A. Eberhardt S. Eisenreich W. Fischer M. Herz S. Illarionov B. et al.Biosynthesis of riboflavin.Vitam. Horm. 2001; 61: 1-49Crossref PubMed Google Scholar, 11Bacher A. Eberhardt S. Fischer M. Kis K. Richter G. Biosynthesis of vitamin B2 (riboflavin).Annu. Rev. Nutr. 2000; 20: 153-167Crossref PubMed Scopus (231) Google Scholar). The phosphorylation of the ribose moiety of riboflavin by riboflavin kinases leads to formation of FMN, whereas FAD is formed via the FAD synthase–catalyzed adenylylation of FMN with ATP as the adenylyl donor (10Bacher A. Eberhardt S. Eisenreich W. Fischer M. Herz S. Illarionov B. et al.Biosynthesis of riboflavin.Vitam. Horm. 2001; 61: 1-49Crossref PubMed Google Scholar, 12McCormick D.B. The intracellular localization, partial purification, and properties of flavokinase from rat liver.J. Biol. Chem. 1962; 237: 959-962Abstract Full Text PDF Google Scholar, 13Sandoval F.J. Roje S. An FMN hydrolase is fused to a riboflavin kinase homolog in plants.J. Biol. Chem. 2005; 280: 38337-38345Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Sandoval F.J. Zhang Y. Roje S. Flavin nucleotide metabolism in plants.J. Biol. Chem. 2008; 283: 30890-30900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). FAD pyrophosphatase, which cleaves AMP from FAD to form FMN, and FMN hydrolases, which release inorganic phosphate from FMN to form riboflavin, are also involved in the interconversion of these flavins (14Sandoval F.J. Zhang Y. Roje S. Flavin nucleotide metabolism in plants.J. Biol. Chem. 2008; 283: 30890-30900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 15Kumar S.A. Rao N.A. Vaidyanathan C.S. Nucleotidases in plants: I. Partial purification and properties of the enzyme hydrolyzing flavine adenine dinucleotide from mung bean seedlings (Phaseolus radiatus).Arch. Biochem. Biophys. 1965; 111: 646-652Crossref PubMed Scopus (13) Google Scholar, 16Byrd J.C. Fearney F.J. Kim Y.S. Rat intestinal nucleotide-sugar pyrophosphatase. Localization, partial purification, and substrate specificity.J. Biol. Chem. 1985; 260: 7474-7480Abstract Full Text PDF PubMed Google Scholar, 17Barile M. Brizio C. De Virgilio C. Delfine S. Quagliariello E. Passarella S. 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An FMN hydrolase is fused to a riboflavin kinase homolog in plants.J. Biol. Chem. 2005; 280: 38337-38345Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Sandoval F.J. Zhang Y. Roje S. Flavin nucleotide metabolism in plants.J. Biol. Chem. 2008; 283: 30890-30900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). However, only a subset of the responsible catalysts have been identified and characterized (5Roje S. Vitamin B biosynthesis in plants.Phytochemistry. 2007; 68: 1904-1921Crossref PubMed Scopus (139) Google Scholar, 13Sandoval F.J. Roje S. An FMN hydrolase is fused to a riboflavin kinase homolog in plants.J. Biol. Chem. 2005; 280: 38337-38345Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Sandoval F.J. Zhang Y. Roje S. Flavin nucleotide metabolism in plants.J. Biol. Chem. 2008; 283: 30890-30900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 19Maruta T. Yoshimoto T. Ito D. Ogawa T. Tamoi M. Yoshimura K. et al.An Arabidopsis FAD pyrophosphohydrolase, AtNUDX23, is involved in flavin homeostasis.Plant Cell Physiol. 2012; 53: 1106-1116Crossref PubMed Scopus (28) Google Scholar, 20Rawat R. Sandoval F.J. Wei Z. Winkler R. Roje S. An FMN hydrolase of the haloacid dehalogenase superfamily is active in plant chloroplasts.J. Biol. Chem. 2011; 286: 42091-42098Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Two plastidially localized Arabidopsis thaliana FAD synthetases, AtRibF1 and AtRibF2, with high homology to each other have previously been reported (14Sandoval F.J. Zhang Y. Roje S. Flavin nucleotide metabolism in plants.J. Biol. Chem. 2008; 283: 30890-30900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Two additional flavin-interconverting enzymes from plastids have been characterized: the FMN hydrolase AtcpFHy1 (20Rawat R. Sandoval F.J. Wei Z. Winkler R. Roje S. An FMN hydrolase of the haloacid dehalogenase superfamily is active in plant chloroplasts.J. Biol. Chem. 2011; 286: 42091-42098Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and the FAD pyrophosphatase ATNUDX23 (19Maruta T. Yoshimoto T. Ito D. Ogawa T. Tamoi M. Yoshimura K. et al.An Arabidopsis FAD pyrophosphohydrolase, AtNUDX23, is involved in flavin homeostasis.Plant Cell Physiol. 2012; 53: 1106-1116Crossref PubMed Scopus (28) Google Scholar, 21Ogawa T. Yoshimura K. Modulation of the subcellular levels of redox cofactors by Nudix hydrolases in chloroplasts.Environ. Exp. Bot. 2019; 161: 57-66Crossref Scopus (4) Google Scholar, 22Ogawa T. Yoshimura K. Miyake H. Ishikawa K. Ito D. Tanabe N. et al.Molecular characterization of organelle-type nudix hydrolases in Arabidopsis.Plant Physiol. 2008; 148: 1412-1424Crossref PubMed Scopus (81) Google Scholar). Of the cytosolic enzymes, only the bifunctional enzyme AtFMN/FHy, which possesses riboflavin kinase and FMN hydrolase activities, has been described (13Sandoval F.J. Roje S. An FMN hydrolase is fused to a riboflavin kinase homolog in plants.J. Biol. Chem. 2005; 280: 38337-38345Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To date, no additional flavin-interconverting enzymes have been characterized in plants. Although the Arabidopsis thaliana FAD synthetases, AtRibF1 and AtRibF2, are sequence homologs to RibC from Bacillus subtilis and RibF from Escherichia coli (14Sandoval F.J. Zhang Y. Roje S. Flavin nucleotide metabolism in plants.J. Biol. Chem. 2008; 283: 30890-30900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the other eukaryotic FAD synthetases identified to date do not share homology to prokaryotic enzymes (23Brizio C. Galluccio M. Wait R. Torchetti E.M. Bafunno V. Accardi R. et al.Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.Biochem. Biophys. Res. Commun. 2006; 344: 1008-1016Crossref PubMed Scopus (64) Google Scholar, 24Galluccio M. Brizio C. Torchetti E.M. Ferranti P. Gianazza E. Indiveri C. et al.Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase.Protein Expr. Purif. 2007; 52: 175-181Crossref PubMed Scopus (37) Google Scholar, 25Pedrolli D.B. Nakanishi S. Barile M. Mansurova M. Carmona E.C. Lux A. et al.The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase.Biochem. Pharmacol. 2011; 82: 1853-1859Crossref PubMed Scopus (39) Google Scholar, 26Wu M. Repetto B. Glerum D.M. Tzagoloff A. Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.Mol. Cell. Biol. 1995; 15: 264-271Crossref PubMed Scopus (77) Google Scholar). Furthermore, while all FAD synthetases described in prokaryotes, with the exception of one from Methanocaldococcus jannaschii, are bifunctional, possessing also riboflavin kinase activity (27Mashhadi Z. Xu H. Grochowski L.L. White R.H. Archaeal RibL: a new FAD synthetase that is air sensitive.Biochemistry. 2010; 49: 8748-8755Crossref PubMed Scopus (22) Google Scholar, 28Efimov I. Kuusk V. Zhang X. McIntire W.S. Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli.Biochemistry. 1998; 37: 9716-9723Crossref PubMed Scopus (60) Google Scholar, 29Mack M. van Loon A.P. Hohmann H.P. Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC.J. Bacteriol. 1998; 180: 950-955Crossref PubMed Google Scholar, 30Manstein D.J. Pai E.F. Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes.J. Biol. Chem. 1986; 261: 16169-16173Abstract Full Text PDF PubMed Google Scholar, 31Kearney E.B. Goldenberg J. Lipsick J. Perl M. Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins.J. Biol. Chem. 1979; 254: 9551-9557Abstract Full Text PDF PubMed Google Scholar), eukaryotic FAD synthetases characterized to date all lack the riboflavin kinase activity (14Sandoval F.J. Zhang Y. Roje S. Flavin nucleotide metabolism in plants.J. Biol. Chem. 2008; 283: 30890-30900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 16Byrd J.C. Fearney F.J. Kim Y.S. Rat intestinal nucleotide-sugar pyrophosphatase. Localization, partial purification, and substrate specificity.J. Biol. Chem. 1985; 260: 7474-7480Abstract Full Text PDF PubMed Google Scholar, 23Brizio C. Galluccio M. Wait R. Torchetti E.M. Bafunno V. Accardi R. et al.Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.Biochem. Biophys. Res. Commun. 2006; 344: 1008-1016Crossref PubMed Scopus (64) Google Scholar, 24Galluccio M. Brizio C. Torchetti E.M. Ferranti P. Gianazza E. Indiveri C. et al.Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase.Protein Expr. Purif. 2007; 52: 175-181Crossref PubMed Scopus (37) Google Scholar, 25Pedrolli D.B. Nakanishi S. Barile M. Mansurova M. Carmona E.C. Lux A. et al.The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase.Biochem. Pharmacol. 2011; 82: 1853-1859Crossref PubMed Scopus (39) Google Scholar, 26Wu M. Repetto B. Glerum D.M. Tzagoloff A. Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.Mol. Cell. Biol. 1995; 15: 264-271Crossref PubMed Scopus (77) Google Scholar, 32Yamada Y. Merrill A.H. McCormick D.B. Probable reaction mechanisms of flavokinase and FAD synthetase from rat liver.Arch. Biochem. Biophys. 1990; 278: 125-130Crossref PubMed Scopus (63) Google Scholar). In addition, the eukaryotic riboflavin kinases characterized to date lack FAD synthetase activity, consistent with a separation of the two activities (13Sandoval F.J. Roje S. An FMN hydrolase is fused to a riboflavin kinase homolog in plants.J. Biol. Chem. 2005; 280: 38337-38345Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 33Santos M.A. Jiménez A. Revuelta J. Molecular characterization of FMN1, the structural gene for the monofunctional flavokinase of Saccharomyces cerevisiae.J. Biol. Chem. 2000; 275: 28618-28624Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 34Karthikeyan S. Zhou Q. Mseeh F. Grishin N.V. Osterman A.L. Zhang H. Crystal structure of human riboflavin kinase reveals a β barrel fold and a novel active site arch.Structure. 2003; 11: 265-273Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A single FAD synthetase, FAD1, has been identified in the model yeast Saccharomyces cerevisiae (26Wu M. Repetto B. Glerum D.M. Tzagoloff A. Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.Mol. Cell. Biol. 1995; 15: 264-271Crossref PubMed Scopus (77) Google Scholar). The activity of this essential enzyme (35Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Véronneau S. et al.Functional profiling of the Saccharomyces cerevisiae genome.Nature. 2002; 418: 387-391Crossref PubMed Scopus (3352) Google Scholar) is dependent on Mg2+ (26Wu M. Repetto B. Glerum D.M. Tzagoloff A. Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.Mol. Cell. Biol. 1995; 15: 264-271Crossref PubMed Scopus (77) Google Scholar). Humans and other animals possess a sequence homolog with a substantial, up to 298 residue, extra domain on the N terminus (36Lynch J.H. Sa N. Saeheng S. Raffaelli N. Roje S. Characterization of a non-nudix pyrophosphatase points to interplay between flavin and NAD(H) homeostasis in Saccharomyces cerevisiae.PLoS One. 2018; 13e0198787Crossref Scopus (5) Google Scholar), which was recently identified as an FAD pyrophosphatase (37Giancaspero T.A. Galluccio M. Miccolis A. Leone P. Eberini I. Iametti S. et al.Human FAD synthase is a bi-functional enzyme with a FAD hydrolase activity in the molybdopterin binding domain.Biochem. Biophys. Res. Commun. 2015; 465: 443-449Crossref PubMed Scopus (23) Google Scholar, 38Leone P. Galluccio M. Brizio C. Barbiroli A. Iametti S. Indiveri C. et al.The hidden side of the human FAD synthase 2.Int. J. Biol. Macromol. 2019; 138: 986-995Crossref PubMed Scopus (16) Google Scholar). The human protein is present as at least two transcript variants, both of which have been shown to also encode Mg2+-dependent enzymes, localized in mitochondria and cytosol, respectively (23Brizio C. Galluccio M. Wait R. Torchetti E.M. Bafunno V. Accardi R. et al.Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.Biochem. Biophys. Res. Commun. 2006; 344: 1008-1016Crossref PubMed Scopus (64) Google Scholar, 24Galluccio M. Brizio C. Torchetti E.M. Ferranti P. Gianazza E. Indiveri C. et al.Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase.Protein Expr. Purif. 2007; 52: 175-181Crossref PubMed Scopus (37) Google Scholar, 25Pedrolli D.B. Nakanishi S. Barile M. Mansurova M. Carmona E.C. Lux A. et al.The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase.Biochem. Pharmacol. 2011; 82: 1853-1859Crossref PubMed Scopus (39) Google Scholar, 39Torchetti E.M. Brizio C. Colella M. Galluccio M. Giancaspero T.A. Indiveri C. et al.Mitochondrial localization of human FAD synthetase isoform 1.Mitochondrion. 2010; 10: 263-273Crossref PubMed Scopus (58) Google Scholar). For isoform 2 only, it has been shown that Co2+ can replace the Mg2+ with only a small reduction in activity (40Torchetti E.M. Bonomi F. Galluccio M. Gianazza E. Giancaspero T.A. Iametti S. et al.Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins.FEBS J. 2011; 278: 4434-4449Crossref PubMed Scopus (47) Google Scholar). Several other FAD synthetases have been purified and characterized from a multitude of species, including M. jannaschii, Corynebacterium ammoniagenes, B. subtilis, Brevibacterium ammoniagenes, and rat (27Mashhadi Z. Xu H. Grochowski L.L. White R.H. Archaeal RibL: a new FAD synthetase that is air sensitive.Biochemistry. 2010; 49: 8748-8755Crossref PubMed Scopus (22) Google Scholar, 28Efimov I. Kuusk V. Zhang X. McIntire W.S. Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli.Biochemistry. 1998; 37: 9716-9723Crossref PubMed Scopus (60) Google Scholar, 31Kearney E.B. Goldenberg J. Lipsick J. Perl M. Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins.J. Biol. Chem. 1979; 254: 9551-9557Abstract Full Text PDF PubMed Google Scholar, 41Oka M. McCormick D.B. Complete purification and general characterization of FAD synthetase from rat liver.J. Biol. Chem. 1987; 262: 7418-7422Abstract Full Text PDF PubMed Google Scholar). All but one have a functional dependence on Mg2+ (28Efimov I. Kuusk V. Zhang X. McIntire W.S. Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli.Biochemistry. 1998; 37: 9716-9723Crossref PubMed Scopus (60) Google Scholar, 31Kearney E.B. Goldenberg J. Lipsick J. Perl M. Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins.J. Biol. Chem. 1979; 254: 9551-9557Abstract Full Text PDF PubMed Google Scholar, 41Oka M. McCormick D.B. Complete purification and general characterization of FAD synthetase from rat liver.J. Biol. Chem. 1987; 262: 7418-7422Abstract Full Text PDF PubMed Google Scholar). The sole exception is an archaeal FAD synthetase from M. jannaschii, which was reported to have maximum activity with Co2+ (27Mashhadi Z. Xu H. Grochowski L.L. White R.H. Archaeal RibL: a new FAD synthetase that is air sensitive.Biochemistry. 2010; 49: 8748-8755Crossref PubMed Scopus (22) Google Scholar). Since catalytic activity with other metal cofactors is not always tested, the ability of these enzymes to substitute another metal for magnesium cannot be ruled out. Here, we report the cloning, characterization, and cytosolic localization of an enzyme from A. thaliana with sequence homology to the FAD synthetases from animals and yeast. The complementary DNA (cDNA) for this enzyme was cloned and recombinantly expressed. The protein product, named AtFADS1, was purified and characterized as the first FAD synthetase having a strong preference for Zn2+ as the associated metal. We also show that the large 243-residue C-terminal domain of the protein, which is homologous to known FAD pyrophosphatases, is not required for the FAD synthetase activity. We also report an apparent lack of FAD pyrophosphatase activity of this enzyme in higher plants, though this activity is present in early diverging lineages, and discuss the implications of such a finding. We previously reported bioinformatic evidence that the gene At5g03430 from A. thaliana encodes a sequence homolog of the S. cerevisiae FAD synthetase FAD1 (13Sandoval F.J. Roje S. An FMN hydrolase is fused to a riboflavin kinase homolog in plants.J. Biol. Chem. 2005; 280: 38337-38345Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Because of the high homology to S. cerevisiae FAD1, we hereafter refer to the protein encoded by gene At5g03430 as AtFADS1. In addition to the homologous region, the putative A. thaliana protein also possesses a substantial, 252-residue, C-terminal extra domain that is homologous to the S. cerevisiae FAD pyrophosphatase FPY1 (YMR178w) (Fig. S1). A broader search reveals that FAD1 homologs are conserved across the entire plant kingdom, invariably with the C-terminal fusion to an FPY1-like domain (Fig. 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 (36Lynch J.H. Sa N. Saeheng S. Raffaelli N. Roje S. Characterization of a non-nudix pyrophosphatase points to interplay between flavin and NAD(H) homeostasis in Saccharomyces cerevisiae.PLoS One. 2018; 13e0198787Crossref Scopus (5) Google Scholar). These contrasting architectures across multiple kingdoms of life are indicative of convergent evolution toward fusion of the two proteins. The presence of two orientations across the different kingdoms reveals two distinct genetic fusion events in evolutionary history, one in an ancestor to humans and other animals, and a separate event in a progenitor to all modern plants, including green algae. Phylogenetic analysis of similar proteins across diverse species demonstrates that the plant proteins form a distinct clade from those in animals, consistent with this proposed evolutionary trajectory (Fig. S2). This convergent evolution is consistent with conserved function of the individual protein domains across the kingdoms, suggesting that AtFADS1 may be a bifunctional FAD synthetase/FAD pyrophosphatase. Preliminary attempts at expressing AtFADS1 in bacterial systems were unsuccessful, resulting in formation of inclusion bodies, so instead expression in yeast was pursued. The cDNAs for full-length AtFADS1 and the truncated protein having just the FAD synthetase domain (AtFADS1trunc) were cloned by reverse transcription–PCR using mRNA isolated from A. thaliana stems as template. Resulting cDNA fragments were subcloned into yeast expression vector pYES-DEST52, followed by functional expression in S. cerevisiae. The purified tagged proteins were used in all subsequent work. Recombinant AtFADS1 was assayed for FAD synthetase activity using a variety of metal activators. Consistent with previously characterized FAD synthetases, a divalent metal cation was necessary for activity (Fig. 1). However, while multiple tested metals were capable of sustaining FAD synthetase activity, the enzyme displayed a strong preference for Zn2+, which yielded a specific activity threefold higher than any other tested metal (Fig. 1). This marks the first time an FAD synthetase has been characterized with preference for zinc ions for maximum activity. Substrate response curves of AtFADS1 for both FMN and ATP show Michaelis–Menten kinetics, though with substrate inhibition by FMN (Fig. 2). Substrate inhibition had previously been reported for AtRibF1 and AtRibF2, the plastidial Arabidopsis FAD synthetases that lack homology to AtFADS1 (14Sandoval F.J. Zhang Y. Roje S. Flavin nucleotide metabolism in plants.J. Biol. Chem. 2008; 283: 30890-30900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and for FAD synthetase from C. ammoniagenes (28Efimov I. Kuusk V. Zhang X. McIntire W.S. Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli.Biochemistry. 1998; 37: 9716-9723Crossref PubMed Scopus (60) Google Scholar), but not from B. subtilis (31Kearney E.B. Goldenberg J. Lipsick J. Perl M. Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins.J. Biol. Chem. 1979; 254: 9551-9557Abstract Full Text PDF PubMed Google Scholar), rat (41Oka M. McCormick D.B. Complete purification and general characterization of FAD synthetase from rat liver.J. Biol. Chem. 1987; 262: 7418-7422Abstract Full Text PDF PubMed Google Scholar), or human (23Brizio C. Galluccio M. Wait R. Torchetti E.M. Bafunno V. Accardi R. et al.Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.Biochem. Biophys. Res. Commun. 2006; 344: 1008-1016Crossref PubMed Scopus (64) Google Scholar, 24Galluccio M. Brizio C. Torchetti E.M. Ferranti P. Gianazza E. Indiveri C. et al.Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase.Protein Expr. Purif. 2007; 52: 175-181Crossref PubMed Scopus (37) Google Scholar, 25Pedrolli D.B. Nakanishi S. Barile M. Mansurova M. Carmona E.C. Lux A. et al.The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase.Biochem. Pharmacol. 2011; 82: 1853-1859Crossref PubMed Scopus (39) Google Scholar). The observed substrate inhibition is consistent with previous proposals that FAD synthetases follow an ordered bi–bi reaction mechanism in which ATP binds prior to FMN (28Efimov I. Kuusk V. 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