title: Dual catalytic activity of hydroxycinnamoyl-coenzyme A quinate transferase from tomato allows it to moonlight in the synthesis of both mono- and dicaffeoylquinic acids.
abstract: Tomato (Solanum lycopersicum), like other Solanaceous species, accumulates high levels of antioxidant caffeoylquinic acids, which are strong bioactive molecules and protect plants against biotic and abiotic stresses. Among these compounds, the monocaffeoylquinic acids (e.g. chlorogenic acid [CGA]) and the dicaffeoylquinic acids (diCQAs) have been found to possess marked antioxidative properties. Thus, they are of therapeutic interest both as phytonutrients in foods and as pharmaceuticals. Strategies to increase diCQA content in plants have been hampered by the modest understanding of their biosynthesis and whether the same pathway exists in different plant species. Incubation of CGA with crude extracts of tomato fruits led to the formation of two new products, which were identified by liquid chromatography-mass spectrometry as diCQAs. This chlorogenate:chlorogenate transferase activity was partially purified from ripe fruit. The final protein fraction resulted in 388-fold enrichment of activity and was subjected to trypsin digestion and mass spectrometric sequencing: a hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT) was selected as a candidate protein. Assay of recombinant HQT protein expressed in Escherichia coli confirmed its ability to synthesize diCQAs in vitro. This second activity (chlorogenate:chlorogenate transferase) of HQT had a low pH optimum and a high Km for its substrate, CGA. High concentrations of CGA and relatively low pH occur in the vacuoles of plant cells. Transient assays demonstrated that tomato HQT localizes to the vacuole as well as to the cytoplasm of plant cells, supporting the idea that in this species, the enzyme catalyzes different reactions in two subcellular compartments.
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.).
authors.forename: Andrea
authors.initials: A
authors.lastname: Moglia
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.).
authors.forename: Sergio
authors.initials: S
authors.lastname: Lanteri
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.).
authors.forename: Cinzia
authors.initials: C
authors.lastname: Comino
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.).
authors.forename: Lionel
authors.initials: L
authors.lastname: Hill
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.).
authors.forename: Daniel
authors.initials: D
authors.lastname: Knevitt
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.).
authors.forename: Cecilia
authors.initials: C
authors.lastname: Cagliero
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.).
authors.forename: Patrizia
authors.initials: P
authors.lastname: Rubiolo
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.).
authors.forename: Stephen
authors.initials: S
authors.lastname: Bornemann
authors.affiliation: Department of Agriculture, Forest and Food Sciences, Plant Genetics and Breeding, University of Torino, 10095 Grugliasco, Italy (A.M., S.L., C.Co.);John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (L.H., D.K., S.B., C.M.); andDepartment of Drug Science and Technology, University of Torino, 10125 Turin, Italy (C.Ca., P.R.) cathie.martin@jic.ac.uk.
authors.forename: Cathie
authors.identifier: http://orcid.org/0000-0002-3640-5080
authors.initials: C
authors.lastname: Martin
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002726:Chlorogenic Acid / Q000737:chemistry / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011801:Quinic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D016415:Sequence Alignment
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; C472707:caffeoylquinic acid; D011801:Quinic Acid; D002726:Chlorogenic Acid; D000217:Acyltransferases; C021394:hydroxycinnamoyl-CoA-quinate transferase; D003065:Coenzyme A