title: Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi.
abstract: Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Shivakumar P
authors.initials: SP
authors.lastname: Devaiah
authors.affiliation: Natural Products Utilization Research Unit, ARS, U.S. Department of Agriculture , P.O. Box 1848, University, Mississippi 38677, United States.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Owens
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Mebrahtu B
authors.initials: MB
authors.lastname: Sibhatu
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Tapasree Roy
authors.initials: TR
authors.lastname: Sarkar
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Christy L
authors.initials: CL
authors.lastname: Strong
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Venkata K P S
authors.initials: VK
authors.lastname: Mallampalli
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Josephat
authors.initials: J
authors.lastname: Asiago
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Cooke
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Starla
authors.initials: S
authors.lastname: Kiser
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Zhangfan
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Anye
authors.initials: A
authors.lastname: Wamucho
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Deborah
authors.initials: D
authors.lastname: Hayford
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Williams
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Peri
authors.initials: P
authors.lastname: Loftis
authors.affiliation: Functional Foods Research Unit, ARS, U.S. Department of Agriculture , Peoria, Illinois 61604, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Berhow
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.
authors.forename: Lee M
authors.initials: LM
authors.lastname: Pike
authors.affiliation: Department of Biological Sciences, East Tennessee State University , P.O. Box 70703, Johnson City, Tennessee 37614, United States.|School of Graduate Studies, East Tennessee State University , P.O. Box 70720, Johnson City, Tennessee 37614, United States.
authors.forename: Cecilia A
authors.initials: CA
authors.lastname: McIntosh
mesh_terms: D000595:Amino Acid Sequence; D032083:Citrus paradisi / Q000201:enzymology*; D003374:Coumarins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics; D005964:Glucosyltransferases / Q000032:analysis* / Q000235:genetics*; D036701:Limonins / Q000378:metabolism; D008969:Molecular Sequence Data; D010636:Phenols / Q000378:metabolism; D010802:Phylogeny; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics*; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D005419:Flavonoids; D036701:Limonins; D010636:Phenols; D011994:Recombinant Proteins; C030123:coumarin; D005964:Glucosyltransferases
keywords: Citrus paradisi; flavonoid; flavor; glucosyltransferase; heterologous expression