Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii
Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii
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DOI：
10.1007/s11033-010-0106-2
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发表时间：
2011-01-01
影响因子：
2.8
通讯作者：
Lee, Ping-Du
中科院分区：
文献类型：
--
作者：
Hsieh, Lu-Sheng;Hsieh, Yi-Lin;Lee, Ping-Du
苯丙氨酸解氨酶是苯丙素途径的第一种酶。从绿竹(Bambusa Oldhamii)cDNA文库中克隆了一个PAL基因，命名为BoPAL1。BoPAL1的开放阅读框全长2,139个核苷酸，编码712个氨基酸的多肽。BoPAL1是被子植物中发现的第一个无内含子PAL基因。在Tail-PCR方法获得的BoPAL1基因5‘侧翼序列中，发现了几个与光反应有关的顺式作用元件，如P box、GT-1基序和SOLIPs。在巴斯德毕赤酵母中表达的重组BoPAL1蛋白具有活性。BoPAL1酶活的最适温度为50℃，最适pH为9.0。凝胶过滤层析测得重组BoPAL1的相对分子质量为323 kDa，全长Bopal的相对分子质量约为80 kDa，表明BoPAL1为同源四聚体。L-Phe的BoPAL1的K(M)和k(CAT)值分别为1.01 mm和10.11 S(-1)。该重组蛋白具有与其他植物中报道的PALS相似的生化性质。
Phenylalanine ammonia-lyase is the first enzyme of general phenylpropanoid pathway. A PAL gene, designated as BoPAL1, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL1 was 2,139 bp in size and predicted to encode a 712-amino acid polypeptide. BoPAL1 was the first intronless PAL gene found in angiosperm plant. Several putative cis-acting elements such as P box, GT-1motif, and SOLIPs involved in light responsiveness were found in the 5'-flanking sequence of BoPAL1 which was obtained by TAIL-PCR method. Recombinant BoPAL1 protein expressed in Pichia pastoris was active. The optimum temperature and pH for BoPAL1 activity was 50A degrees C and 9.0, respectively. The molecular mass of recombinant BoPAL1 was estimated as 323 kDa using gel filtration chromatography and the molecular mass of full-length BoPAL was about 80 kDa, indicating that BoPAL1 presents as a homotetramer. The K (m) and k (cat) values of BoPAL1 for L-Phe were 1.01 mM and 10.11 s(-1), respectively. The recombinant protein had similar biochemical properties with PALs reported in other plants.