Characterization of tocopherol cyclases from higher plants and cyanobacteria. Evolutionary implications for tocopherol synthesis and function

Abstract

Tocopherols are lipophilic antioxidants synthesized exclusively by photosynthetic organisms and collectively constitute vitamin E, an essential nutrient for both humans and animals. Tocopherol cyclase (TC) catalyzes the conversion of various phytyl quinol pathway intermediates to their corresponding tocopherols through the formation of the chromanol ring. Herein, the molecular and biochemical characterization of TCs from Arabidopsis (VTE1 [VITAMIN E 1]), Zea mays (SXD1 [Sucrose Export Deficient 1]) and Synechocystis sp. PCC6803 (slr1737) are described. Mutations in the VTE1, SXD1, or slr1737 genes resulted in both tocopherol deficiency and the accumulation of 2,3-dimethyl-6-phytyl-1,4-benzoquinone (DMPBQ), a TC substrate. Recombinant SXD1 and VTE1 proteins are able to convert DMPBQ to gamma-tocopherol in vitro. In addition, expression of maize SXD1 in a Synechocystis sp. PCC6803 slr1737 knockout mutant restored tocopherol synthesis, indicating that TC activity is evolutionarily conserved between plants and cyanobacteria. Sequence analysis identified a highly conserved 30-amino acid C-terminal domain in plant TCs that is absent from cyanobacterial orthologs. vte1-2 causes a truncation within this C-terminal domain, and the resulting mutant phenotype suggests that this domain is necessary for TC activity in plants. The defective export of Suc in sxd1 suggests that in addition to presumed antioxidant activities, tocopherols or tocopherol breakdown products also function as signal transduction molecules, or, alternatively, the DMPBQ that accumulates in sxd1 disrupts signaling required for efficient Suc export in maize.

Figure 1.

Tocopherol biosynthetic pathway. This figure represents the enzymatic reactions and intermediates that are involved in tocopherol synthesis. 1, HPPD. 2, Homogentisate phytyl transferase (HPT). 3, 2-Methyl-6-phytyl-1,4-benzoquinone (MPBQ) methyltransferase. 4, TC. 5, γ-Tocopherol methyltransferase (γ-TMT). HPP, p-Hydroxyphenylpyruvate; HGA, homogentisic acid; SAM, S-adenosyl l-Met.

Figure 2.

HPLC analysis of tocopherols in wild-type and mutant Arabidopsis, maize, and Synechocystis sp. PCC6803. Tocopherols present in Arabidopsis, maize, and Synechocystis sp. PCC6803 lipid extracts were separated by normal phase HPLC and detected using a fluorescence detector with 290-nm excitation and 325-nm emission. Tocol, a synthetic tocopherol, was used as an internal recovery standard. A, Arabidopsis leaf tissue: solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. B, Maize leaf tissue: solid line, wild type; dotted line, sxd1. C, Synechocystis sp. PCC6803: solid line, wild type; dotted line, Δslr1737 insertional mutant; gray line, SXD1 expressed in the Δslr1737 insertional mutant. Retention times of α-, β-, δ-, and γ-tocopherol and tocol were determined by HPLC analysis of tocopherol standards. LU, Luminescence units.

Figure 3.

HPLC analysis of the prenyl quinones from wild-type and mutant Arabidopsis, maize, and Synechocystis sp. PCC6803. Lipids were extracted from Arabidopsis, maize, and Synechocystis sp. PCC6803, and total prenyl quinines were isolated by thin-layer chromatography (TLC) and then analyzed by normal phase HPLC (see “Materials and Methods”) A, Arabidopsis. Solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. B, Maize. Solid line, Wild type; dotted line, sxd1. C, Synechocystis sp. PCC6803. Solid line, Wild type; dotted line, Δslr1737 insertional mutant; gray line, SXD1cDNA expressed in the Δslr1737 mutant background. Insets, Spectra of the peak labeled DMPBQ. Phyllo, Phylloquinone; PQ, Plastoquinone.

Figure 4.

HPLC analysis of seed tocopherols in wild-type Arabidopsis, vte1-1, and vte1-2. Total seed lipids were extracted, and the tocopherols present were separated by reverse phase HPLC and detected using a fluorescence detector; 290-nm excitation and 325-nm emission. Tocol, a synthetic tocopherol, was used as an internal recovery standard. Solid line, Columbia wild type; dotted line, vte1-1; gray line, vte1-2. Retention times of α-, δ-, and γ-tocopherol and tocol were determined by HPLC analysis of tocopherol standards.

Figure 5.

Alignment of the carboxy termini of TC orthologs from plants and cyanobacteria. The asterisk above the At4g32770 protein sequence denotes the position of the vte1-2 mutation. Anabaena, Anabaena sp. PCC7120 (all0245); Chlamy, Chlamydomonas reinhardtii; Medicago, M. truncatula; Nostoc, N. punctiforme (506-74); Physcomitrella, Physcomitrella patens; Synecho, Synechococcus sp. PCC7002. The P. patens, rice (Oryza sativa), and wheat (Triticum aestivum) sequences are partial sequences obtained from ESTs. Dark shading, Amino acid identity; light shading, Amino acid similarity. The threshold for amino acid consensus identity or similarity is 51%.

Figure 6.

TC activity of proteins expressed in E. coli. E. coli cell lysates from cells overexpressing the empty pET vector or pET engineered to express TC proteins from Arabidopsis, maize, and Synechocystis sp. PCC6803 were incubated with radiolabeled 2,3-methyl-6-phytyl-1,4-benzonequinol (3 methyl ¹⁴C) for 4 h as described in “Materials and Methods.” Total lipids were extracted, separated by TLC, and radiolabeled products were detected by phosphor imager analysis. Products were identified by comigration with standards. The ¹⁴C incorporation into γ-tocopherol was quantified densitometrically and expressed as pixels per microgram of total protein.