Universität Wien, Fakultät für Chemie, Institut für Biophysikalische Chemie, Wien, Austria.
Sci Rep. 2019 Mar 11;9(1):4022. doi: 10.1038/s41598-019-39687-0.
Polyphenol oxidases (PPOs) contain the structurally similar enzymes tyrosinases (TYRs) and catechol oxidases (COs). Two cDNAs encoding pro-PPOs from tomato (Solanum lycopersicum) were cloned and heterologously expressed in Escherichia coli. The two pro-PPOs (SlPPO1-2) differ remarkably in their activity as SlPPO1 reacts with the monophenols tyramine (k = 7.94 s) and phloretin (k = 2.42 s) and was thus characterized as TYR, whereas SlPPO2 accepts only diphenolic substrates like dopamine (k = 1.99 s) and caffeic acid (k = 20.33 s) rendering this enzyme a CO. This study, for the first time, characterizes a plant TYR and CO originating from the same organism. Moreover, X-ray structure analysis of the latent holo- and apo-SlPPO1 (PDB: 6HQI and 6HQJ) reveals an unprecedented high flexibility of the gatekeeper residue phenylalanine (Phe270). Docking studies showed that depending on its orientation the gatekeeper residue could either stabilize and correctly position incoming substrates or hinder their entrance into the active site. Furthermore, phloretin, a substrate of SIPPO1 (K = 0.11 mM), is able to approach the active centre of SlPPO1 with both phenolic rings. Kinetic and structural results indicate that phloretin could act as a natural substrate and connote the participation of PPOs in flavonoid-biosynthesis.
多酚氧化酶（PPOs）包含结构相似的酶酪氨酸酶（TYRs）和儿茶酚氧化酶（COs）。从番茄（Solanum lycopersicum）克隆并异源表达了两个编码前体 PPO 的 cDNA。这两个前体 PPO（SlPPO1-2）在活性上差异显著，SlPPO1 与单酚类化合物酪胺（k=7.94s）和根皮苷（k=2.42s）反应，因此被鉴定为 TYR，而 SlPPO2 仅接受二酚类底物，如多巴胺（k=1.99s）和咖啡酸（k=20.33s），使其成为 CO。本研究首次从同一生物体中鉴定出植物 TYR 和 CO。此外，潜伏态全酶和脱辅基酶 SlPPO1 的 X 射线结构分析（PDB：6HQI 和 6HQJ）揭示了看门残基苯丙氨酸（Phe270）前所未有的高灵活性。对接研究表明，看门残基的取向可以稳定并正确定位进入的底物，或者阻止它们进入活性中心。此外，SlPPO1 的底物根皮苷（K=0.11mM）能够使两个酚环都接近 SlPPO1 的活性中心。动力学和结构结果表明，根皮苷可能作为一种天然底物发挥作用，并暗示 PPOs 参与类黄酮生物合成。
