Table_1_Characterization of Arabidopsis thaliana Hydroxyphenylpyruvate Reductases in the Tyrosine Conversion Pathway.DOCX

Tyrosine serves as a precursor to several types of plant natural products of medicinal or nutritional interests. Hydroxyphenylpyruvate reductase (HPPR), which catalyzes the reduction of 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), has been shown to be the key enzyme in the biosynthesis of rosmarinic acid (RA) from tyrosine and, so far, HPPR activity has been reported only from the RA-accumulating plants. Here, we show that HPPR homologs are widely distributed in land plants. In Arabidopsis thaliana, which does not accumulate RA at detectable level, two homologs (HPPR2 and HPPR3) are functional in reducing pHPP. Phylogenetic analysis placed HPPR2 and HPPR3 in two separate groups within the HPPR clade, and HPPR2 and HPPR3 are distinct from HPR1, a peroxisomal hydroxypyruvate reductase (HPR). In vitro characterization of the recombinant proteins revealed that HPPR2 has both HPR and HPPR activities, whereas HPPR3 has a strong preference for pHPP, and both enzymes are localized in the cytosol. Arabidopsis mutants defective in either HPPR2 or HPPR3 contained lower amounts of pHPL and were impaired in conversion of tyrosine to pHPL. Furthermore, a loss-of-function mutation in tyrosine aminotransferase (TAT) also reduced the pHPL accumulation in plants. Our data demonstrate that in Arabidopsis HPPR2 and HPPR3, together with TAT1, constitute to a probably conserved biosynthetic pathway from tyrosine to pHPL, from which some specialized metabolites, such as RA, can be generated in specific groups of plants. Our finding may have broad implications for the origins of tyrosine-derived specialized metabolites in general.

酪氨酸是多种兼具药用与营养学价值的植物天然产物的前体。羟苯基丙酮酸还原酶（Hydroxyphenylpyruvate reductase, HPPR）可催化4-羟苯基丙酮酸（4-hydroxyphenylpyruvic acid, pHPP）还原为4-羟苯基乳酸（4-hydroxyphenyllactic acid, pHPL），该酶被证实是从酪氨酸合成迷迭香酸（rosmarinic acid, RA）的关键酶；截至目前，HPPR活性仅在积累迷迭香酸的植物中被报道。本研究发现，HPPR同源蛋白广泛分布于陆生植物中。在无法检测到迷迭香酸积累的拟南芥（Arabidopsis thaliana）中，存在HPPR2与HPPR3两个同源蛋白，二者均可催化pHPP的还原反应。系统发育分析显示，HPPR2与HPPR3分别隶属于HPPR进化枝的两个独立分支，且二者均不同于过氧化物酶体羟丙酮酸还原酶（peroxisomal hydroxypyruvate reductase, HPR1）。对重组蛋白的体外表征结果表明，HPPR2同时具备HPR与HPPR活性，而HPPR3对pHPP具有极强的底物偏好性，且两种酶均定位于细胞质基质中。分别缺失HPPR2或HPPR3的拟南芥突变体，其体内pHPL含量降低，且酪氨酸向pHPL的转化过程受损。此外，酪氨酸转氨酶（tyrosine aminotransferase, TAT）的功能丧失突变也会降低植物体内的pHPL积累水平。本研究数据证实，在拟南芥中，HPPR2、HPPR3与TAT1共同构成了一条从酪氨酸到pHPL的保守生物合成途径，特定植物类群可通过该途径合成迷迭香酸等特化代谢产物。本研究发现对于解析酪氨酸衍生特化代谢物的整体起源具有广泛的参考价值。