VITAMIN E BIOSYNTHESIS: BIOCHEMICAL CHARACTERIZATION OF BARLEY HOMOGENTISIC ACID GERANYLGERANYL DIPHOSPHATE TRANSFERASE

Authors: Hunter, Sarah; Cahoon, Edgar

Submitted to: National Plant Lipid Cooperative Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/31/2005
Publication Date: 6/10/2005
Citation: Hunter, S.C., Cahoon, E.B. 2005. Vitamin e biosynthesis: biochemical characterization of barley homogentisic acid geranylgeranyl diphosphate transferase [abstract]. National Plant Lipid Cooperative Meeting. p. 33.

Interpretive Summary:

Technical Abstract: Tocotrienols and tocopherols, both vitamin E compounds, consist of an aromatic head group and a lipophilic tail. Both function as antioxidants in plants and human diets. Tocotrienols are found mainly in monocot seeds, while tocopherols are the principle vitamin E found in leaves and most dicot seeds. The first committed step of vitamin E biosynthesis is the condensation of homogentisate with a prenyl diphosphate - either the polyunsaturated geranylgeranyl diphosphate (GGDP) in the case of tocotrienols, or the saturated phytyl diphosphate (PDP), in the case of tocopherols. We have recently identified the enzyme termed “homogentisate geranylgeranyl transferase” (HGGT) that catalyzes the initial step of tocotrienol biosynthesis in monocot seeds. HGGTs from rice, wheat, and barley share < 50% amino acid sequence identity with known homogentisate phytyltransferases (HPTs), which catalyze the analogous initial reaction for tocopherol biosynthesis. Secondary structure predictions for both HGGTs and HPTs indicate seven transmembrane regions and place these enzymes in the UbiA prenyltransferase family. This family contains a 23 amino acid signature motif which is a proposed active site. However, in HGGTs and HPTs, only the 11 N-terminal amino acids of the signature are present. HGGT from barley endosperm (HvHGGT) and HPT from Arabidopsis (AtHPT) were expressed in Sf-21 insect cells. Membrane-enriched extracts from these cells had prenyltransferase activity, while uninfected cells did not. Relative activity with GGDP was four to five-fold higher than with PDP in HvHGGT-expressing cells. In contrast, AtHPT-expressing cells produced approximately 50- to 80-fold more product when PDP, rather than GGDP, was used as a prenyl donor. Thus, HvHGGT and AtHPT are biochemically distinct enzymes. We are currently exploring the structural basis for the different substrate specificities of these enzymes by domain swapping and site-directed mutagenesis approaches.