Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana

Abstract

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(1H,3H) pyrimidinedione 5'-phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (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 AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar K values for the substrate ARPP, with AtcpFHy/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.

An overview on the identification of MAIT cell antigens

“…Taken together, these experiments definitively pinpointed the bacterial metabolite necessary to generate activating MAIT cell ligands as 5‐A‐RU. These studies have now been recapitulated for E. coli and S. pneumoniae by other groups, and also demonstrate the requirement for ribD/G in these bacteria …”

“…A further complication is that the enzyme nomenclature also differs among bacteria. The details of riboflavin synthesis and its regulation are complex and still emerging, with the recent discovery of the 5‐A‐RU 5′‐phosphate phosphatase in Arabidopsis thaliana, completing the pathway.…”

The novel phosphatase RosC catalyzes the last unknown step of roseoflavin biosynthesis in Streptomyces davaonensis

“…(Haase et al, 2013;Sarge et al, 2015). The protein AtPyrP2 from Arabidopsis thaliana was found to be very specific for ARPP and silencing of the AtPyrP2 gene led to decreased levels of riboflavin, RP and FAD (Sa et al, 2016). This finding suggests that in plants a riboflavin pathway-specific ARPP phosphatase is present.…”

Production of riboflavin and related cofactors by biotechnological processes

“…A study from 2011 showed that an FMN-specific hydrolase (AtcpFHy1) from the HAD superfamily was able to catalyze the dephosphorylation of FMN [46]. A few years later, in 2016, the plastidic FMN hydrolase AtcpFHy1 (At1g79790) together with the gene products of At4g11570 and At4g25840, were identified as having ArPP phosphatase activity [47]. In 2015, the HAD superfamily phosphatase EFI-501083 from Bacteroides thetaiotaomicron (B. thetaiotaomicron) was predicted to catalyze the dephosphorylation of ArPP according to covalent docking models [48].…”