Mapping Enzymatic Esterification to Natural Expression Levels for a Specialized Clade of HCT Acyltransferases in Poplar
Authors:
Brian G. Fox1,2* ([email protected]), Rebecca A. Smith1, Justin Acheson2, Craig A. Bingman1,2, Hailey R. Sieren2, Ella C. Lodewyck2, Ella R. Torkelson2, Noah Kaufman3, Kirk Vander Meulen1,2, Vitaliy Tymokhin1, Steven D. Karlen1, Shawn D. Mansfield1,3, John Ralph1,2
Institutions:
1Great Lakes Bioenergy Research Center, University of Wisconsin–Madison; 2Department of Biochemistry, University of Wisconsin–Madison; 3Department of Wood Science, University of British Columbia, Vancouver, Canada
Goals
Hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferases (HCT) are an important group of BAHD acyltransferase enzymes because of their key roles in the lignin biosynthetic pathway and the biosynthesis of plant specialized metabolites, such as chlorogenic acid (caffeoylquinic acid) and p-coumaroyl shikimate. The inherent promiscuity of the BAHD acyltransferases leads to a combinatorial array of plant natural products and offers interesting targets for both enzyme and plant engineering. Many plants have large HCT families, but the differences in substrate specificity are not well understood.
Abstract
To address this knowledge gap, researchers screened all Populus trichocarpa BAHD acyltransferases for HCT activity and found a distinct clade of nine enzymes with various activities. Results show two distinct catalytic classes of HCTs in poplar residing in different branches of the clade; shikimate-specific enzymes (HSTs), likely involved in lignin biosynthesis based on expression data, and quinate-preferring enzymes (HQTs). The extent of substrate promiscuity and competitive preferences of the different enzymes were also determined. Both the HST and HQT enzymes were found to generate shikimate or quinate ester products and convert the products back into CoA thioester and acid substrates under appropriate reaction conditions. Active site residues potentially involved in switching the reaction specificity between HSTs and HQTs were identified through AlphaFold protein structure analysis and tested for their role in defining HCT substrate specificity and activity by site-directed mutagenesis. Insights from this work will be presented.
Funding Information
This work was supported by the U.S. DOE, Office of Science, Basic Energy Sciences under award #DE-SC0020349 and #DE-SC0023013 to B.G.F. R.A.S., C.A.B., K.V.M, S.D.K., V.T., S.D.M., and J.R. were supported, in part, by the Great Lakes Bioenergy Research Center (GLBRC, DOE BER Office of Science DE-SC0018409). This study made use of the MS and NMR facilities at the GLBRC.