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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Functional Foods Research » Research » Publications at this Location » Publication #307086
Title: Identification, recombinant expression, and biochemical analysis of putative secondary product glucosyltransferases from Citrus paradisi

Author
item DEVAIAH, SHIVAKUMAR - East Tennessee State University
item Owens, Daniel
item SIBHATU, MEBRAHTU - East Tennessee State University
item SARKAR, TAPASREE - East Tennessee State University
item STRONG, CHRISTY - East Tennessee State University
item MALLAMPALLI, VENKATA K P S. - East Tennessee State University
item ASIAGO, JOSEPHAT - East Tennessee State University
item COOKE, JENNIFER - East Tennessee State University
item KAISER, STARLA - East Tennessee State University
item LIN, ZHANGFAN - East Tennessee State University
item WAMUCHO, ANYE - East Tennessee State University
item HAYFORD, DEBORAH - East Tennessee State University
item WILLIAMS, BRUCE - East Tennessee State University
item LOFTIS, PERI - East Tennessee State University
item Berhow, Mark
item PIKE, LEE - East Tennessee State University
item MCINTOSH, CECILIA - East Tennessee State University

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2016
Publication Date: 2/18/2016
Citation: Devaiah, S.P., Owens, D.K., Sibhatu, M.B., Sarkar, T.R., Strong, C.L., Mallampalli, V., Asiago, J., Cooke, J., Kaiser, S., Lin, Z., Wamucho, A., Hayford, D., Williams, B.E., Loftis, P., Berhow, M.A., Pike, L.M., Mcintosh, C.A. 2016. Identification, recombinant expression, and biochemical analysis of putative secondary product glucosyltransferases from Citrus paradisi. Journal of Agricultural and Food Chemistry. 64(9): 1957-1969. https://doi.org/10.1021/acs.jafc.5b05430.
DOI: https://doi.org/10.1021/acs.jafc.5b05430

Interpretive Summary: Secondary product enzymes that add sugars to natural product compounds found in plants are involved in the accumulation of many of the naturally occurring compounds found in plants. Flavonoid and limonoid glycosides impact taste properties of many Citrus species, especially grapefruit. The search for the genes that control the express of these sugar transferase may result in a better understanding of how these genes could be manipulated to provide better tasting fruit. Nine secondary product glucose transferase genes were obtained and screened from grapefruit. They were cloned and expressed and assessed for activity with a variety of flavonoid and limonoid substrates and three were shown to have glucose transferase activity in the enzyme assay, although the activities varied greatly depanding on the assay buffer conditions. These results provide valuable information to the field of secondary product glucosyltransferase structure and function.

Technical 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.