Phenol-oxidizing laccases from the termite gut
Phenol-oxidizing laccases from the termite gut
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DOI：
10.1016/j.ibmb.2010.07.004
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发表时间：
2010-10-01
影响因子：
3.8
通讯作者：
Scharf, M. E.
中科院分区：
文献类型：
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作者：
Coy, M. R.;Salem, T. Z.;Scharf, M. E.
对黄足散白蚁（Reticulitermes flavipes）的两种肠漆酶同工型（RfLacA和RfLacB）的cDNA进行了测序。系统发育分析比较翻译的R。flavipes漆酶与其他67种原核和真核生物漆酶的比较结果表明，R. flavipes漆酶在进化上是独特的。与晶体学验证的漆酶的比对证实，参与金属结合的肽基序在两种异构体中是100%保守的。漆酶转录和酚氧化酶活性最丰富的共生体自由唾液腺和前肠组织，验证的基因和活动的主机派生。使用杆状病毒-昆虫表达系统，这两种亚型与组氨酸标签一起功能性表达，并纯化至接近同质。RflacA的ICP-MS（电感耦合等离子体-质谱法）分析鉴定了主要由铜组成的结合金属（类似于每个漆酶蛋白分子4个铜分子，并且类似于每个组氨酸标签3个铜分子），以及较少量的钙、锰和锌。两种重组酶制剂对木质素单体芥子酸和其他四种酚类底物表现出较强的活性。相比之下，这两种亚型显示出对四种黑色素前体的低得多的活性或没有活性，表明这两种亚型都不参与珠被形成。还观察到重组RfLacA制剂对木质素碱的修饰。这些发现为R.黄足菌肠漆酶在进化上是不同的、宿主来源的、在唾液腺中产生、分泌到前肠中、结合铜并在木质纤维素消化中起作用。这些发现有助于更好地了解白蚁消化和肠道生理学，并将有助于未来的翻译研究，研究个别白蚁酶在木质纤维素消化的贡献。(C)2010爱思唯尔有限公司版权所有。
cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforrns. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RflacA identified bound metals consisting mainly of copper (similar to 4 copper molecules per laccase protein molecule and similar to 3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion. (C) 2010 Elsevier Ltd. All rights reserved.