Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

Abstract

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiaminrequiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470. Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content

“…Since this is the first plant protein to exhibit this type of enzymatic activity, we will name this enzyme ThMPase1. However, knockouts of ThMPase1 in Arabidopsis have not revealed any visible phenotype or modification of thiamin concentration, even when combined with other mutant lines affected in thiamin monophosphate content Mimura et al, 2016).In this report, we show that PPsPase1 and PECP1 (but not ThMPase1) constitute robust and sensitive reporters of Pi status, indicating a very dynamic expression response to Pi concentration. Using a reverse genetics approach, we altered the expression of these proteins and revealed that PPsPase1 and PECP1 are likely involved in the recycling of polar heads from membrane lipids, but do not play a role in the overall regulation of membrane lipid composition or plant responses to Pi starvation.…”

“…Since this is the first plant protein to exhibit this type of enzymatic activity, we will name this enzyme ThMPase1. However, knockouts of ThMPase1 in Arabidopsis have not revealed any visible phenotype or modification of thiamin concentration, even when combined with other mutant lines affected in thiamin monophosphate content Mimura et al, 2016).…”

On the nature of thiamine triphosphate in Arabidopsis

“…., 2015). By contrast, the pale green 1 (pale1) mutant, impaired in the TMP phosphatase, TH2 (At5g32470), is deficient in leaf TDP (Mimura et al, 2016, Hsieh et al, 2017. We examined the B 1 vitamer profile of these lines and could confirm that TDP was increased in THIC THI1 OE but decreased in pale1 ( Figure 3B).…”

Genome‐wide identification of EMBRYO‐DEFECTIVE (EMB) genes required for growth and development in Arabidopsis

“…Conversely, seedling defects have been noted for weak alleles of several EMB genes listed in Table 4 (Conklin et al, 2000;Song et al, 2004;Noutoshi et al, 2005;Mo et al, 2006). Seedling mutant phenotypes have also been documented for Arabidopsis auxotrophs altered in eight additional genes required for the biosynthesis of thiamine (Komeda et al, 1988;Papini-Terzi et al, 2003;Raschke et al, 2007;Mimura et al, 2016), tryptophan (Last & Fink, 1988), riboflavin (Ouyang et al, 2010;Hedtke et al, 2012), and isoleucine (Yu et al, 2013). Seedling defects in the trp1 auxotroph are not rescuable by tryptophan (Last & Fink, 1988) because other intermediates in tryptophan biosynthesis are required for auxin-mediated plant growth and development.…”