title: Identification and characterization of the missing pyrimidine reductase in the plant riboflavin biosynthesis pathway.
abstract: Riboflavin (vitamin B₂) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.
authors.affiliation: Department of Horticultural Sciences, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: Ghulam
authors.initials: G
authors.lastname: Hasnain
authors.forename: Océane
authors.initials: O
authors.lastname: Frelin
authors.forename: Sanja
authors.initials: S
authors.lastname: Roje
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Ellens
authors.forename: Kashif
authors.initials: K
authors.lastname: Ali
authors.forename: Jiahn-Chou
authors.initials: JC
authors.lastname: Guan
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Garrett
authors.forename: Valérie
authors.initials: V
authors.lastname: de Crécy-Lagard
authors.forename: Jesse F
authors.initials: JF
authors.lastname: Gregory
authors.forename: Donald R
authors.initials: DR
authors.lastname: McCarty
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Hanson
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D060365:Chloroplast Proteins / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics; D019295:Computational Biology / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017343:Genes, Plant; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009709:Nucleotide Deaminases / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics; D010766:Phosphorylation; D010802:Phylogeny; D021381:Protein Transport; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012256:Riboflavin / Q000096:biosynthesis*; D016415:Sequence Alignment; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D060365:Chloroplast Proteins; D029968:Escherichia coli Proteins; D011743:Pyrimidines; D011994:Recombinant Proteins; D009249:NADP; D010088:Oxidoreductases; D013401:Sugar Alcohol Dehydrogenases; C514675:PHS1 protein, Arabidopsis; D017027:Protein Tyrosine Phosphatases; C104887:RibD protein, E coli; D009709:Nucleotide Deaminases; C030986:pyrimidine; D012256:Riboflavin