Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii
Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii
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DOI：
10.1016/j.phytochem.2010.09.019
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发表时间：
2010-12-01
期刊：
影响因子：
3.8
通讯作者：
Lee, Ping-Du
中科院分区：
文献类型：
--
作者：
Hsieh, Lu-Sheng;Ma, Guo-Jhang;Lee, Ping-Du
青竹中的苯丙氨酸解氨酶（PAL，EC 4 3 1 5）是从竹子壳中分离和克隆的。竹壳 PAL 的 (L)-Phe 的 Km 为 476 RM，天然 PAL 的分子量估计为 275 kDa，亚基的分子量约为 76 kDa，表明来自竹子的 PAL 也以四聚体形式存在。PAL 活性的最适温度为50 ℃，最佳 pH 90 使用 Q-TOF 串联 MS/MS 从头测序确认纯化竹壳 PAL 的身份 从 B oldhamii 中克隆了 4 个 PAL 基因，命名为 B0PAL1 至 B0PAL4 B0PAL3 和 B0PAL4 的开放阅读框大小分别为 2142 和 2106 bp B0PAL2-4 包含 1 个内含子和 2 个外显子，但未发现内含子大肠杆菌中表达的 BoPAL1 B0PAL4 具有 PAL 和酪氨酸解氨酶活性。虽然重组野生型 PAL 蛋白与天然竹壳 PAL 具有相似的生化特性，但 B0PAL1 F133H 和 B0PAL2 F134H 的定点诱变分别显示出针对 L-Phe 的 k(cat)/K-m 值降低，而 B0PAL2 F134H 的 k(cat)/K-m 值略有增加L-Tyr 的 k(cat)/K-m 值 这些数据表明其他残基在很大程度上控制 Phe/Tyr 底物特异性 针对纯化壳 PAL 产生的抗体用于组织化学研究 在竹壳和枝芽中 PAL 主要定位于厚壁组织细胞 (C) 2010 Elsevier Ltd 保留所有权利
Phenylalanine ammonia-lyase (PAL, EC 4 3 1 5) from green bamboo was Isolated and cloned from the shell of Bambusa oldhamii The Km of bamboo shell PAL for (L)-Phe was 476 RM and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa indicating that PAL from bamboo also exists as a tetramer The optimum temperature for PAL activity was 50 degrees C and the optimal pH 90 The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing Four PAL genes designated as B0PAL1 to B0PAL4 were cloned from B oldhamii The open reading frames of B0PAL3 and B0PAL4 were 2142 and 2106 bp in size respectively B0PAL2-4 contained one intron and two exons but no intron was found in BoPAL1 B0PAL4 expressed in Escherichia cob possessed both PAL and tyrosine ammonia-lyase activities While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of B0PAL1 F133H and B0PAL2 F134H respectively showed decreased k(cat)/K-m values toward L-Phe whereas B0PAL2 F134H showed a slightly increased k(cat)/K-m value toward L-Tyr These data suggest other residues largely control Phe/Tyr substrate specificity An antibody raised against the purified shell PAL was generated for histochemical studies In bamboo shell and branch shoots PAL was localized primarily in sclerenchyma cells (C) 2010 Elsevier Ltd All rights reserved