綠竹苯丙胺酸脫氨裂解酶之生化學與分子生物學研究

Abstract

苯丙胺酸脫氨裂解酶 (Phenylalanine ammonia-lyase, PAL, EC 4.3.1.5 or 4.3.1.24) 催化苯丙胺酸 (phenylalanine) 非氧化型脫氨反應產生肉桂酸 (trans-cinnamic acid)，是植物 phenylpropanoids 生合成的第一個關鍵酵素。許多植物二級代謝產物為 phenylpropanoids 衍生物，如黃酮素 (flavonoids)、花青素 (anthocyanins)、植物荷爾蒙、木質素 (lignin) 與植物殺菌素 (phytoalexins) 等。 綠竹 PAL 研究以竹殼蛋白質純化與基因選殖等方法進行。以 Q-TOF 質譜法鑑定純化的 PAL 之胺酸序列。篩選綠竹 cDNA 庫得到兩條 PAL 基因，命名為 BoPAL1 與 BoPAL2，ORF 分別為 2,139 bp 與 2,142 bp，彼此有 91% 核苷酸相似度與 96% 胺酸相似度。從綠竹 cDNA 中以 PCR 方法分離出全長 BoPAL3 與 BoPAL4 及不完整之 BoPAL5 基因。BoPAL2、BoPAL3 與 BoPAL4 皆由一個 intron 與兩個 exons 組成，但 BoPAL1 沒有 intron 存在。綠竹 PAL 胺酸具有保守性之活性區序列，Ala-Ser-Gly 三個胺酸會自體催化成 3,5-dihydro-5-methylidine-H-imidazol-4-one (MIO) 輔基。BoPALs 基因表現在 Escherichia coli 與 Pichia pastories 中，BoPAL1 與 BoPAL2 都以同質四元體存在，且可以偵測到 PAL 活性，重組蛋白質生化性質與原態竹殼 PAL 類似。大腸桿菌表現之 BoPAL2 重組蛋白質作為免疫小白鼠的抗原，製備之抗體可以專一性辨識原態與重組 PAL 蛋白質，使用 anti-PAL 抗體進行免疫組織化學研究。綠竹維管束組織 (vascular bundle) 中 PAL 主要定位於厚壁細胞 (sclerenchyma cells) 內，PAL 會參與木質素的生合成，與維管束組織發育相關。以 TAIL-PCR 成功選殖 BoPALs 基因的 5’-端上游區域，帶有可能之 TATA box 與數個 MRE (MYB recognition elements)。

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5 or 4.3.1.24) catalyzes the non-oxidative deamination of phenylalanine to trans-cinnamic acid and ammonia, the first step in the biosynthesis of phenylpropanoids. Many secondary metabolic products in plants, such as flavonoids, anthocyanin, plant hormone, lignin, phytoalexin, and benzoic acid derivatives, are derived from phenylpropanoids. PAL from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Two PAL genes, designated as BoPAL1 and BoPAL2, were cloned from a Bambusa oldhamii cDNA library. The open reading frames of BoPAL1 and BoPAL2 were 2,139 and 2,142 bp in size, respectively. Three cDNAs of two full-length BoPAL3, BoPAL4 and a partialBoPAL5 sequences were isolated from bamboo by PCR-based cloning. BoPAL2, BoPAL3 and BoPAL4 consisted of one intron and two exons, but no intron was found in BoPAL1. BoPAL1 showed 91% nucleotide sequence identity and 96% protein sequence identity with BoPAL2. All PAL sequences contained a conserved active site motif, Ala-Ser-Gly triad, which can be converted into a 3,5-dihydro-5-methylidine-H-imidazol-4-one (MIO) prosthetic group. Both BoPAL1 and BoPAL2 genes were expressed in Escherichia coli and Pichia pastories. The recombinant proteins exhibited PAL activity and also existed as a homotetramer. The recombinant proteins had similar biochemical properties to the native bamboo shell PAL. Antibody against recombinant E. coli-expressed BoPAL2 was generated for immunohistochemical studies. The anti-PAL antibody could specifically recognize the protein isolated from bamboo and the recombinant proteins. In the vascular bundles of bamboo tissues, PAL was localized primarily in the sclerenchyma cells, which are lignified cells. During the development of vascular bundles, PAL enzyme in sclerenchyma cells would participate in lignin biosynthesis. Using a modified TAIL-PCR technique, the 5’-flanking region of the two BoPAL genes were successfully isolated. In the region isolated, a putative TATA box and several MRE (MYB recognition elements) could be identified.