RESUMO
For more than 80 years, tocopherols have been known to be an essential nutrient, vitamin E, for humans and animals. Work in recent years has concentrated on dissecting tocopherol biosynthesis to engineer the pathway in agricultural crops. Molecular dissection of the pathway in plants is now complete with the cloning and characterization of the gene for Arabidopsis MPBQ/MSBQ methyltransferase (VITAMIN E 3; VTE3). Alison Van Eennemaan and colleagues used seed-specific expression of two vitamin E pathway methyltransferases to engineer increased vitamin E activity in soybeans.
Assuntos
Arabidopsis/genética , Produtos Agrícolas/genética , Engenharia Genética/métodos , Glycine max/genética , Vitamina E/biossíntese , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Produtos Agrícolas/enzimologia , Metiltransferases/genética , Metiltransferases/metabolismo , Glycine max/enzimologia , Glycine max/metabolismo , Tocoferóis/metabolismo , Vitamina E/químicaRESUMO
Tocopherols are lipid-soluble compounds synthesized only by photosynthetic eukaryotes and oxygenic cyanobacteria. The pathway and enzymes for tocopherol synthesis are homologous in cyanobacteria and plants except for 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ MT), which catalyzes a key methylation step in both tocopherol and plastoquinone (PQ) synthesis. Using a combined genomic, genetic, and biochemical approach, we isolated and characterized the VTE3 (vitamin E defective) locus, which encodes MPBQ/MSBQ MT in Arabidopsis. The phenotypes of vte3 mutants are consistent with the disruption of MPBQ/MSBQ MT activity to varying extents. The ethyl methanesulfonate-derived vte3-1 allele alters tocopherol composition but has little impact on PQ levels, whereas the null vte3-2 allele is deficient in PQ and alpha- and gamma-tocopherols. In vitro enzyme assays confirmed that VTE3 is the plant functional equivalent of the previously characterized MPBQ/MSBQ MT (Sll0418) from Synechocystis sp PCC6803, although the two proteins are highly divergent in primary sequence. Sll0418 orthologs are present in all fully sequenced cyanobacterial genomes, Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana but absent from vascular and nonvascular plant databases. VTE3 orthologs are present in all vascular and nonvascular plant databases and in C. reinhardtii but absent from cyanobacterial genomes. Intriguingly, the only prokaryotic genomes that contain VTE3-like sequences are those of two species of archea, suggesting that, in contrast to all other enzymes of the plant tocopherol pathway, the evolutionary origin of VTE3 may have been archeal rather than cyanobacterial. In vivo analyses of vte3 mutants and the corresponding homozygous Synechocystis sp PCC6803 sll0418::aphII mutant revealed important differences in enzyme redundancy, the regulation of tocopherol synthesis, and the integration of tocopherol and PQ biosynthesis in cyanobacteria and plants.
Assuntos
Arabidopsis/enzimologia , Cianobactérias/enzimologia , Metiltransferases/genética , Metiltransferases/metabolismo , Fotossíntese/genética , Plastoquinona/metabolismo , Tocoferóis/metabolismo , Sequência de Aminoácidos , Arabidopsis/classificação , Arabidopsis/genética , Sequência de Bases , Sequência Conservada , Cianobactérias/classificação , Cianobactérias/genética , Metiltransferases/química , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
A putative 2-methyl-6-phytylbenzoquinone (MPBQ) methyltransferase gene, SLL0418, was identified from the Synechocystis PCC6803 genome based on its homology to previously characterized gamma-tocopherol methyltransferases. Genetic and biochemical evidence confirmed open reading frame (ORF) SLL0418 encodes a MPBQ methyltransferase. An SLL0418 partial knockout mutant accumulated beta-tocopherol with no effect in the overall tocopherol content of the cell. In vitro assays of the SLL0418 gene expressed in Escherichia coli showed the enzyme efficiently catalyzes methylation of ring carbon 3 of MPBQ. In addition, the enzyme also catalyzes the methylation of ring carbon 3 of 2-methyl-6-solanylbenzoquinol in the terminal step of plastoquinone biosynthesis.
