Cytosolic Hydroxymethyldihydropterin Pyrophosphokinase/Dihydropteroate Synthase from Arabidopsis thaliana

Abstract

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/ DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction. Functionality of cytHPPK/DHPS was addressed by two parallel approaches: first, the cytHPPK/DHPS was able to rescue yeast mutants lacking corresponding activities; second, recombinant cytHPPK/DHPS expressed and purified from Escherichia coli displayed both HPPK and DHPS activities in vitro. In contrast to mitHPPK/DHPS, which was ubiquitously expressed, the cytHPPK/DHPS gene was exclusively expressed in reproductive tissue, more precisely in developing seeds as revealed by histochemical analysis of a transgenic cytHPPK/DHPS promoter-GUS line. In addition, it was observed that expression of cytHPPK/DHPS mRNA was induced by salt stress, suggesting a potential role of the enzyme in stress response. This was supported by the phenotype of a T-DNA insertion mutant in the cytHPPK/DHPS gene, resulting in lower germination rates as compared with the wild-type upon application of oxidative and osmotic stress.Folates play a crucial role as cofactors in a number of onecarbon transfer reactions. These reactions are essential for DNA synthesis and the methylation cycle occurring in all organisms (for review see Refs.

Does Abiotic Stress Cause Functional B Vitamin Deficiency in Plants?

“…resistance at germination (Storozhenko et al, 2007;Navarrete et al, 2012). (4) Riboflavin: The Arabidopsis phs1 (PHOTOSENSITIVE1) mutant, first identified by its sensitivity to high-light stress (Ouyang et al, 2010), was found to lack a functional domain of the riboflavin biosynthesis enzyme PyrR Frelin et al, 2015).…”

Interplay between Sucrose and Folate Modulates Auxin Signaling in Arabidopsis

“…2A; Prabhu et al, 1997Prabhu et al, , 1998Storozhenko et al, 2007). DHPS is an enzyme that combines dihydropterin and pABA to form dihydropteroate, which is then polyglutamylated and reduced to form THF (Hanson and Gregory, 2002).…”

“…It was hypothesized that if DHPS is the true target of SMX, then elevating DHPS abundance would confer resistance to the compound. The Arabidopsis (Arabidopsis thaliana) genome contains two DHPS isoforms, one mitochondrial (At4g30000) and one cytosolic (At1g69190; Storozhenko et al, 2007); each was constitutively expressed under the control of the Cauliflower mosaic virus 35S promoter in planta. Activity of the construct was confirmed by measuring the expression of DHPS in three independently transformed lines using quantitative PCR (Supplemental Fig.…”

A question of balance: achieving appropriate nutrient levels in biofortified staple crops

“…In this context, the consumption of a folate tracer with a light breakfast meal reduced bioavailability by 15 % compared with the folate tracer alone (111) . Folate bioavailability can be increased by enhancing the activity of g-glutamyl hydrolase in plants, as this would be released from the vacuole following maceration and would promote the release of unconjugated folate from the food matrix before ingestion (110) .…”

“…The monoglutamyl: polyglutamyl folate ratio in the diet appears to have no influence on the efficiency of absorption, suggesting that the enzymic deconjugation is not a rate-limiting step (109) . However, the entrapment of folate in food matrices can influence bioavailability by rendering it inaccessible to the epithelial enzymes (110) . In this context, the consumption of a folate tracer with a light breakfast meal reduced bioavailability by 15 % compared with the folate tracer alone (111) .…”