title: Molecular cloning and functional analysis of the ortho-hydroxylases of p-coumaroyl coenzyme A/feruloyl coenzyme A involved in formation of umbelliferone and scopoletin in sweet potato, Ipomoea batatas (L.) Lam.
abstract: Ortho-hydroxylation of cinnamates is a key step in coumarin biosynthesis in plants. Ortho-hydroxylated cinnamates undergo trans/cis isomerization of the side-chain and then lactonization to form coumarins. Sweet potato [Ipomoea batatas (L.) Lam.] accumulates umbelliferone and scopoletin after biotic and abiotic stresses. To elucidate molecular aspects of ortho-hydroxylation involved in umbelliferone formation in sweet potato, isolation and characterization of cDNAs encoding 2-oxoglutarate-dependent dioxygenases (2OGD) was performed from sweet potato tubers treated with a chitosan elicitor. Five cDNAs (designated as Ib) encoding a protein of 358 amino acid residues were cloned, and these were categorized into two groups, Ib1 and Ib2, based on their amino acid sequences. Whether the recombinant Ib proteins had any enzymatic activity toward cinnamates was examined. Ib1 proteins exhibited ortho-hydroxylation activity toward feruloyl coenzyme A (CoA) to form scopoletin (K(m)=~10 μM, k(cat)=~2.7s(-1)). By contrast, Ib2 proteins catalyzed ortho-hydroxylation of feruloyl-CoA (K(m)=7.3-14.0 μM, k(cat)=0.28-0.55 s(-1)) and also of p-coumaroyl-CoA (K(m)=6.1-15.2 μM, k(cat)=0.28-0.64 s(-1)) to form scopoletin and umbelliferone, respectively. Fungal and chitosan treatments increased levels of umbelliferone and its glucoside (skimmin) in the tubers, and expression of the Ib2 gene was induced concomitantly.
authors.affiliation: Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 6110011, Japan.
authors.forename: Seitaro
authors.initials: S
authors.lastname: Matsumoto
authors.forename: Masaharu
authors.initials: M
authors.lastname: Mizutani
authors.forename: Kanzo
authors.initials: K
authors.lastname: Sakata
authors.forename: Bun-Ichi
authors.initials: B
authors.lastname: Shimizu
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids; D048271:Chitosan; D002934:Cinnamates / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005658:Fungi; D015870:Gene Expression; D017343:Genes, Plant; D006900:Hydroxylation; D027723:Ipomoea batatas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D035281:Plant Tubers / Q000201:enzymology*; D011994:Recombinant Proteins; D012603:Scopoletin / Q000378:metabolism*; D013312:Stress, Physiological; D014468:Umbelliferones / Q000096:biosynthesis*
chemical_list: D000214:Acyl Coenzyme A; D000596:Amino Acids; D002934:Cinnamates; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D014468:Umbelliferones; C119167:feruloyl-CoA; C058644:4-coumaroyl-coenzyme A; C031477:7-hydroxycoumarin; D048271:Chitosan; D006899:Mixed Function Oxygenases; D012603:Scopoletin