An FMN Hydrolase of the Haloacid Dehalogenase Superfamily Is Active in Plant Chloroplasts

Rawat, Renu; Sandoval, Francisco J.; Wei, Zhaoyang; Winkler, Robert; Roje, Sanja

doi:10.1074/jbc.m111.260885

Cited by 21 publications

(31 citation statements)

References 37 publications

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“…We previously confirmed the plastidial localization of AtcpFHy/PyrP1 (Rawat et al ., ), and therefore this enzyme was used as a positive control. Subcellular localization of AtPyrP2 and AtGpp1/PyrP3 was studied using fluorescence microscopy to observe the leaves from stably transformed Arabidopsis.…”

Section: Resultsmentioning

confidence: 99%

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

Rawat

Thornburg

et al. 2016

The Plant Journal

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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.

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Section: Resultsmentioning

confidence: 99%

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

Rawat

Thornburg

et al. 2016

The Plant Journal

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“…The conversion of riboflavin to FMN is catalyzed by riboflavin kinase; we have not yet detected the activity of this enzyme in H. albus roots, although Sandoval and Roje [16] have reported its assay using a recombinant Escherichia coli. These authors have found FMN hydrolase to be present both in the cytosol [16] and in chloroplasts [1]; and in the case of the cytosolic hydrolase, there is fusion of FMN hydrolase (FHy) and riboflavin kinase (RibK) genes (AY878327) [16]. In our present study, we have successfully cloned the corresponding fused cDNA from H. albus (Table 1); and therefore -if both genes are functionalconfirmation of this gene fusion in H. albus relates directly to the possibility of an apparent 'futile cycle', as discussed above.…”

Section: Discussionmentioning

confidence: 98%

“…Although the existence of FMN hydrolase activity in plants has been confirmed based on gene expression and enzyme activity, its physiological relevance remains speculative [1,16]. In the present paper, therefore, we have investigated whether secreted riboflavin originates from the de novo synthesis of riboflavin or, alternatively, from the partial decomposition of FMN.…”

Section: Introductionmentioning

confidence: 97%

“…Plants, in common with other organisms, can biosynthesize riboflavin de novo and then convert it into its nucleotide derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), both of which are usually incorporated into various proteins as co-factors. These flavins mediate electron transfers and are involved in various redox-requiring processes, such as mitochondrial electron transport, photosynthesis, fatty acid oxidation, photoreception, DNA repair and the biosynthesis of various secondary metabolites [1,2]; accordingly, flavins in free form are not usually abundant in ordinary living plant tissues. Interestingly, however, free flavins, especially riboflavin, have been detected in the roots and/or root exudates of some taxonomically unrelated dicotyledonous plants, including tobacco and sunflower, growing under conditions of Fe deficiency [3,4].…”

Section: Introductionmentioning

confidence: 99%

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Increased de novo riboflavin synthesis and hydrolysis of FMN are involved in riboflavin secretion from Hyoscyamus albus hairy roots under iron deficiency

Higa

Khandakar

Mori

et al. 2012

Plant Physiology and Biochemistry

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Riboflavin secretion by Hyoscyamus albus hairy roots under Fe deficiency was examined to determine where riboflavin is produced and whether production occurs via an enhancement of riboflavin biosynthesis or a stimulation of flavin mononucleotide (FMN) hydrolysis. Confocal fluorescent microscopy showed that riboflavin was mainly localized in the epidermis and cortex of the root tip and, at the cellular level, in the apoplast. The expressions of three genes involved in the de novo biosynthesis of riboflavin (GTP cyclohydrolase II/3,6, riboflavin synthase) were compared between Fe-starved and Fe-replete roots over a time course of 7 days, using RT-PCR. All three genes were found to be highly expressed over the period 1-7 days in the roots cultured under Fe deficiency. Since riboflavin secretion began to be detected only from 3 days, there was a lag phase observed between the increased transcript accumulations and riboflavin secretion. To determine whether FMN hydrolysis might contribute to the riboflavin secretion in Fe-deficient root cultures, FMN hydrolase activity was determined and was found to be substantially increased after 3 days, when riboflavin secretion became detectable. These results suggested that not only de novo riboflavin synthesis but also the hydrolysis of FMN contributes to riboflavin secretion under conditions of Fe deficiency. Respiration activity was assayed during the time-course, and was also found to be enhanced after 3 days under Fe deficiency, suggesting a possible link with riboflavin secretion. On the other hand, several respiratory inhibitors were found not to affect riboflavin synthase transcript accumulation.Key words: Hyoscyamus albus; hairy roots; iron deficiency; riboflavin secretion; riboflavin biosynthesis; FMN hydrolase; respiration. RibA: GTP cyclohydrolase II · RibB: 3,4-dihydroxy-2-butanone 4-phosphate synthase · RibC: 6,7-dimethyl-8-ribityllumazine synthase · RibD: riboflavin synthase

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“…The riboflavin biosynthetic pathway appears to proceed through the same steps in plants, yeast, and bacteria, although the enzymes in plants remain to be identified (Fischer et al 2004(Fischer et al , 2005Sandoval and Roje 2005;Sandoval et al 2008;Giancaspero et al 2009;Rawat et al 2011;Maruta et al 2012). E. gracilis Z has been shown to synthesize, store (39.0 ng mg −1 dry weight), and excrete (39 ng ml −1 medium) riboflavin (Baker et al 1981).…”

Section: Vitamin B2mentioning

confidence: 94%

Biochemistry and Physiology of Vitamins in Euglena

Watanabe

Yoshimura

Shigeoka

2017

Advances in Experimental Medicine and Biology

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Euglena gracilis Z requires vitamins B1 and B12 for growth. It takes up and accumulates large amounts of these exogenous vitamins through energy-dependent active transport systems. Except for these essential vitamins, E. gracilis Z has the ability to synthesize all human vitamins. Euglena synthesizes high levels of antioxidant vitamins such as vitamins C and E, and, thus, are used as nutritional supplements for humans and domestic animals. Methods to effectively produce vitamins in Euglena have been investigated.Previous biochemical studies indicated that E. gracilis Z contains several vitamin-related novel synthetic enzymes and metabolic pathways which suggests that it is a highly suitable organism for elucidating the physiological functions of vitamins in comparative biochemistry and biological evolution. E. gracilis Z has an unusual biosynthetic pathway for vitamin C, a hybrid of the pathways found in animals and plants. This chapter presents up-to-date information on the biochemistry and physiological functions of vitamins in this organism.

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An FMN Hydrolase of the Haloacid Dehalogenase Superfamily Is Active in Plant Chloroplasts

Cited by 21 publications

References 37 publications

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

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

Increased de novo riboflavin synthesis and hydrolysis of FMN are involved in riboflavin secretion from Hyoscyamus albus hairy roots under iron deficiency

Biochemistry and Physiology of Vitamins in Euglena

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