Universität Wien, Fakultät für Chemie, Institut für Biophysikalische Chemie, Wien, Austria.
Sci Rep. 2020 Feb 3;10(1):1659. doi: 10.1038/s41598-020-57671-x.
Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono- versus diphenolase activity in the type-III copper enzyme jrPPO1.
多酚氧化酶（PPOs）包括酪氨酸酶（TYRs）和儿茶酚氧化酶（COs），它们催化黑色素生物合成的初始反应。TYRs 羟化单酚（单酚酶活性）和氧化二酚（二酚酶活性）底物，而 COs 仅与二酚反应。为了阐明 PPOs 中不同反应的生化基础，从核桃叶片中合成 cDNA，克隆编码潜伏核桃酪氨酸酶（jrPPO1）的靶基因，并在大肠杆菌中异源表达该酶。针对两个活性控制器残基（Asn240 和 Leu244）和守门员残基（Phe260）设计了突变，以削弱 jrPPO1 的单酚酶活性。首次在两个双突变体（Asn240Lys/Leu244Arg 和 Asn240Thr/Leu244Arg）中阻断了 jrPPO1 对 L-酪氨酸的单酚酶活性，而其二酚酶活性部分保留，从而将 jrPPO1 转化为 CO。动力学数据表明，重组 jrPPO1 类似于天然酶，分光光度研究证明突变对铜含量没有影响。本文提供的结果提供了实验证据，表明位于活性中心周围的氨基酸之间的精确相互作用控制了底物特异性，并由此控制了 III 型铜酶 jrPPO1 中的单酚酶与二酚酶活性。
