Sullivan Michael L, Knollenberg Benjamin J
US Dairy Forage Research Center, USDA-ARS, Madison, WI, United States.
Department of Plant Sciences, Pennsylvania State University, University Park, PA, United States.
Front Plant Sci. 2021 Nov 9;12:727461. doi: 10.3389/fpls.2021.727461. eCollection 2021.
Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2--caffeoyl-L-malate) and clovamide [-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 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 , the encoded proteins were capable of transferring hydroxycinnamic acids (-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 -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 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.
红三叶草叶片积累了高水平（高达干物质的1%至2%）的两种咖啡酸衍生物：phaselic酸（2 - 咖啡酰 - L - 苹果酸）和clovamide [-咖啡酰 - L - 3,4 - 二羟基苯丙氨酸（L - DOPA）]。这些物质可能在保护植物免受生物和非生物胁迫方面发挥作用，但在收获和储存饲料期间，还能通过内源性多酚氧化酶防止蛋白质氧化。我们之前从红三叶草中鉴定并表征了一种羟基肉桂酰辅酶A（CoA）：苹果酸羟基肉桂酰转移酶（HMT）。在此，我们在未展开的红三叶草叶片中鉴定出一种羟基肉桂酰 - CoA：L - DOPA羟基肉桂酰转移酶（HDT）活性。尽管HMT酶缺乏HDT活性，但之前克隆的HMT基因沉默后，红三叶草中的HMT和HDT活性均降低。基于BAHD羟基肉桂酰 - CoA转移酶序列的简并引物与5'和3' cDNA末端快速扩增相结合的PCR方法，用于从红三叶草中克隆出两个几乎相同的cDNA。当在[具体表达宿主未给出]中表达时，编码的蛋白质能够将羟基肉桂酸（对香豆酸、咖啡酸或阿魏酸）从相应的CoA硫酯转移到芳香族氨基酸L - Phe、L - Tyr、L - DOPA或L - Trp上。测定了这些底物的动力学参数。HDT在转基因苜蓿中的稳定表达导致苜蓿叶片积累了苜蓿中通常不存在的对香豆酰 - 和阿魏酰 - L - Tyr，但没有含咖啡酰或L - DOPA部分的衍生物。HDT在[具体表达宿主未给出]中的瞬时表达导致了咖啡酰 - L - Tyr的产生，但没有clovamide。HDT与酪氨酸羟化酶的共表达导致clovamide积累，这表明宿主物种中可用氨基酸（和羟基肉桂酰 - CoA）底物库可能在决定植物中HDT产物积累方面起主要作用。最后，HDT和HMT蛋白具有高度的同一性（72%），但底物特异性有很大差异，这为进一步研究这类酶的结构 - 功能关系带来了希望，这可能允许合理设计具有特定和理想活性的BAHD酶。
