Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides

Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide [N-caffeoyl-L-3,4-dihydroxyphenylalanine (L-DOPA)]. These likely play roles in protecting the plant from biotic and abiotic stresses but can als...

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Autores principales:	Michael L. Sullivan, Benjamin J. Knollenberg
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	BAHD hydroxycinnamoyl-CoA hydroxycinnamoyl transferase clovamide CYP76AD6 hydroxycinnamoyl amide phenylpropanoid red clover Plant culture SB1-1110
Acceso en línea:	https://doaj.org/article/e3c56ab51a134f1ebc5ce9cfc2aa6ff9
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spelling	oai:doaj.org-article:e3c56ab51a134f1ebc5ce9cfc2aa6ff92021-11-19T16:41:01ZRed Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides1664-462X10.3389/fpls.2021.727461https://doaj.org/article/e3c56ab51a134f1ebc5ce9cfc2aa6ff92021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fpls.2021.727461/fullhttps://doaj.org/toc/1664-462XRed clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide [N-caffeoyl-L-3,4-dihydroxyphenylalanine (L-DOPA)]. These likely play 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. We previously identified and characterized, a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT) from red clover. Here, we identified a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) activity in unexpanded red clover leaves. Silencing of the previously cloned HMT gene reduced both HMT and HDT activities in red clover, even though the HMT enzyme lacks HDT activity. 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 thioesters 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 resulted in clovamide accumulation, indicating the host species’ pool of available amino acid (and hydroxycinnamoyl-CoA) substrates likely plays a major role in determining HDT product accumulation in planta. Finally, that HDT and HMT proteins share a high degree of identity (72%), but differ substantially in substrate specificity, is promising for further investigation of structure-function relationships of this class of enzymes, which could allow the rational design of BAHD enzymes with specific and desirable activities.Michael L. SullivanBenjamin J. KnollenbergFrontiers Media S.A.articleBAHD hydroxycinnamoyl-CoA hydroxycinnamoyl transferaseclovamideCYP76AD6hydroxycinnamoyl amidephenylpropanoidred cloverPlant cultureSB1-1110ENFrontiers in Plant Science, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	BAHD hydroxycinnamoyl-CoA hydroxycinnamoyl transferase clovamide CYP76AD6 hydroxycinnamoyl amide phenylpropanoid red clover Plant culture SB1-1110
spellingShingle	BAHD hydroxycinnamoyl-CoA hydroxycinnamoyl transferase clovamide CYP76AD6 hydroxycinnamoyl amide phenylpropanoid red clover Plant culture SB1-1110 Michael L. Sullivan Benjamin J. Knollenberg Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides
description	Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide [N-caffeoyl-L-3,4-dihydroxyphenylalanine (L-DOPA)]. These likely play 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. We previously identified and characterized, a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT) from red clover. Here, we identified a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) activity in unexpanded red clover leaves. Silencing of the previously cloned HMT gene reduced both HMT and HDT activities in red clover, even though the HMT enzyme lacks HDT activity. 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 thioesters 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 resulted in clovamide accumulation, indicating the host species’ pool of available amino acid (and hydroxycinnamoyl-CoA) substrates likely plays a major role in determining HDT product accumulation in planta. Finally, that HDT and HMT proteins share a high degree of identity (72%), but differ substantially in substrate specificity, is promising for further investigation of structure-function relationships of this class of enzymes, which could allow the rational design of BAHD enzymes with specific and desirable activities.
format	article
author	Michael L. Sullivan Benjamin J. Knollenberg
author_facet	Michael L. Sullivan Benjamin J. Knollenberg
author_sort	Michael L. Sullivan
title	Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides
title_short	Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides
title_full	Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides
title_fullStr	Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides
title_full_unstemmed	Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides
title_sort	red clover hdt, a bahd hydroxycinnamoyl-coenzyme a:l-3,4-dihydroxyphenylalanine (l-dopa) hydroxycinnamoyl transferase that synthesizes clovamide and other n-hydroxycinnamoyl-aromatic amino acid amides
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/e3c56ab51a134f1ebc5ce9cfc2aa6ff9
work_keys_str_mv	AT michaellsullivan redcloverhdtabahdhydroxycinnamoylcoenzymeal34dihydroxyphenylalanineldopahydroxycinnamoyltransferasethatsynthesizesclovamideandothernhydroxycinnamoylaromaticaminoacidamides AT benjaminjknollenberg redcloverhdtabahdhydroxycinnamoylcoenzymeal34dihydroxyphenylalanineldopahydroxycinnamoyltransferasethatsynthesizesclovamideandothernhydroxycinnamoylaromaticaminoacidamides
_version_	1718420043009622016

Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides

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