Kinetic analysis of red clover HDT, a hydroxycinnamoyl-CoA:L-DOPA transferase, and production of clovamide and related amides in planta

Authors: Sullivan, Michael; KNOLLENBERG, BENJAMIN - Pennsylvania State University

Submitted to: Plant Biology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/17/2021
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Red clover leaves accumulate high levels (up to approximately 1% of dry matter) of clovamide (N-caffeoyl-L-3,4-dihydroxyphenylalanine [L-DOPA]). This likely plays roles in protecting the plant from biotic and abiotic stresses, but can also help preserve protein during harvest and storage of the forage via oxidation by an endogenous polyphenol oxidase. Clovamide and its derivatives may also have desirable bioactive properties and thus potential nutraceutical and medicinal value. A combination of PCR with degenerate primers based on BAHD hydroxycinnamoyl-CoA transferase sequences and 5' and 3' rapid amplification of cDNA ends was used to clone two nearly identical cDNAs from red clover. When expressed in Escherichia coli, the encoded proteins were capable of transferring hydroxycinnamic acids (p-coumaric, caffeic, or ferulic) from the corresponding CoA thiolesters to the aromatic amino acids L-Phe, L-Tyr, L-DOPA, or L-Trp. Kinetic parameters for these substrates were determined. Stable expression of HDT in transgenic alfalfa resulted in foliar accumulation of p-coumaroyl- and feruloyl-L-Tyr that are not normally present in alfalfa, but not derivatives containing caffeoyl or L-DOPA moieties. Transient expression of HDT in Nicotiana benthamiana resulted in the production of caffeoyl-L-Tyr, but not clovamide. Coexpression of HDT with a tyrosine hydroxylase did result in clovamide accumulation. These findings show that the hydroxycinnamoyl amide products formed by HDT in planta largely depend on the available pool of both hydroxycinnamoyl-CoA donor and amino acid acceptor substrates in conjunction with the efficiency with which HDT can use the various substrates (i.e., its kinetic parameters). Furthermore, if heterologous clovamide biosynthesis is to be successfully engineered in plant species that lack sufficient hydroxylase activity towards either L-Tyr or caffeoyl-L-Tyr (formed by HDT), coexpression of a tyrosine hydroxylase will likely be required.