title: Purification and characterization of a novel galloyltransferase involved in catechin galloylation in the tea plant (Camellia sinensis).
abstract: Catechins (flavan-3-ols), the most important secondary metabolites in the tea plant, have positive effects on human health and are crucial in defense against pathogens of the tea plant. The aim of this study was to elucidate the biosynthetic pathway of galloylated catechins in the tea plant. The results suggested that galloylated catechins were biosynthesized via 1-O-glucose ester-dependent two-step reactions by acyltransferases, which involved two enzymes, UDP-glucose:galloyl-1-O-β-D-glucosyltransferase (UGGT) and a newly discovered enzyme, epicatechin:1-O-galloyl-β-D-glucose O-galloyltransferase (ECGT). In the first reaction, the galloylated acyl donor β-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphosphate glucose. In the second reaction, galloylated catechins were produced by ECGT catalysis from β-glucogallin and 2,3-cis-flavan-3-ol. 2,3-cis-Flavan-3-ol and 1-O-galloyl-β-D-glucose were appropriate substrates of ECGT rather than 2,3-trans-flavan-3-ol and 1,2,3,4,6-pentagalloylglucose. Purification by more than 1641-fold to apparent homogeneity yielded ECGT with an estimated molecular mass of 241 to 121 kDa by gel filtration. Enzyme activity and SDS-PAGE analysis indicated that the native ECGT might be a dimer, trimer, or tetramer of 60- and/or 58-kDa monomers, and these monomers represent a heterodimer consisting of pairs of 36- or 34- of and 28-kDa subunits. MALDI-TOF-TOF MS showed that the protein SCPL1199 was identified. Epigallocatechin and epicatechin exhibited higher substrate affinities than β-glucogallin. ECGT had an optimum temperature of 30 °C and maximal reaction rates between pH 4.0 and 6.0. The enzyme reaction was inhibited dramatically by phenylmethylsulfonyl fluoride, HgCl(2), and sodium deoxycholate.
authors.affiliation: School of Life Science, Ministry of Education in China, Anhui Agricultural University, 130 West Changjiang Rd, Hefei, Anhui 230036, China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Liu
authors.forename: Liping
authors.initials: L
authors.lastname: Gao
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Qin
authors.initials: Q
authors.lastname: Yang
authors.forename: Zhongwei
authors.initials: Z
authors.lastname: Lu
authors.forename: Zhiyin
authors.initials: Z
authors.lastname: Nie
authors.forename: Yunsheng
authors.initials: Y
authors.lastname: Wang
authors.forename: Tao
authors.initials: T
authors.lastname: Xia
mesh_terms: D028241:Camellia sinensis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D002392:Catechin / Q000378:metabolism*; D004795:Enzyme Stability; D005707:Gallic Acid / Q000378:metabolism*; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D005707:Gallic Acid; D002392:Catechin; D014166:Transferases