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Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana
Author(s) -
Sa Na,
Rawat Renu,
Thornburg Chelsea,
Walker Kevin D.,
Roje Sanja
Publication year - 2016
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13291
Subject(s) - flavin mononucleotide , biochemistry , flavin adenine dinucleotide , arabidopsis thaliana , biosynthesis , riboflavin , flavin group , cofactor , enzyme , arabidopsis , biology , phosphatase , plastid , chemistry , chloroplast , gene , mutant
Summary Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide ( FAD ) and flavin mononucleotide ( FMN ), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5‐amino‐6‐ribitylamino‐2,4(1 H ,3 H ) pyrimidinedione 5′‐phosphate ( ARPP ) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase Atcp FH y1 (At1g79790), belonging to the haloacid dehalogenase ( HAD ) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP . AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of Atcp FH y/PyrP1 and AtPyrP2 showed that they have similar K m values for the substrate ARPP , with Atcp FH y/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP . Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0–8.5 and 40–50°C. T‐DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN , and FAD . Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana . The identification of this enzyme closes a long‐standing gap in understanding of the riboflavin biosynthesis in plants.