Arginine kinase from the beetle Cissites cephalotes (Olivier).: Molecular cloning, phylogenetic analysis and enzymatic properties
Arginine kinase from the beetle Cissites cephalotes (Olivier).: Molecular cloning, phylogenetic analysis and enzymatic properties
复制标题
DOI：
10.1016/j.ibmb.2006.12.006
复制
发表时间：
2007-04-01
影响因子：
3.8
通讯作者：
Suzuki, Tomohiko
中科院分区：
文献类型：
--
作者：
Tanaka, Kunuko;Ichinari, Shuichi;Suzuki, Tomohiko
在这里，我们报告的PCR扩增和克隆精氨酸激酶（AK）的cDNA从甲虫Cissites cephalotes（Olivier）。cDNA全长1210 bp，开放阅读框1125 bp，5'和3'非翻译区分别为30和55 bp。开放阅读框编码374个氨基酸的蛋白质，其中大部分残基被认为是AK功能所必需的：预测与底物精氨酸相互作用的5个残基（S77、Y82、E239、C285和ED 28），以及预测与底物ADP相互作用的5个残基（R138、R140、R243、R294和R323）。节肢动物AKs的系统发育树表明，昆虫AKs可分为两大类：一类是来自不同种类昆虫的典型AKs，另一类是从基因组序列推测的AKs序列。Cissites AK聚集在第2组，并提供了第一个证据表明，第2组基因确实在昆虫中表达。此外，我们在大肠杆菌中表达了顺式AK蛋白作为与麦芽糖结合蛋白的融合物，并测定了正向反应的动力学常数（K-m，K-d，V-max和k（cat））。与其他来源（昆虫、软体动物和棘皮动物）的AKs的动力学常数的比较表明，来自Cissites和Periplaneta的昆虫AKs具有两个非常独特的特征，在AKs中最低的k（cat）（和k（cat）/K-m（arg）），以及缺乏协同底物结合（K-d/K-m近似于1）。(c)2007爱思唯尔有限公司保留所有权利。
Here, we report the PCR amplification and cloning of a cDNA for arginine kinase (AK) from the beetle Cissites cephalotes (Olivier). The cDNA is 1210 bp and has an open reading frame of 1125 bp and 5' and 3'-untranslated regions of 30 and 55 bp, respectively. The open reading frame encodes a 374 amino acid protein with most of the residues considered necessary for AK function: five residues predicted to interact with the substrate arginine (S77, Y82, E239, C285 and ED28), and five residues predicted to interact with the substrate ADP (R138, R140, R243, R294 and R323). A phylogenetic tree of arthropod AKs indicated clearly that insect AKs can be separated into typical AKs from various insect species (group 1) and putative AK sequences deduced from genomic sequences (group 2). Cissites AK clustered in group 2 and provides the first evidence that a group-2 gene is indeed expressed in insects. Moreover, we expressed Cissites AK protein in Escherichia coli as a fusion with maltose-binding protein, and kinetic constants (K-m, K-d, V-max and k(cat)) were determined for the forward reaction. Comparison of kinetic constants with those of AKs from other sources (insects, mollusks and echinoderms) indicated that insect AKs from Cissites and Periplaneta have two very unique features, the lowest k(cat) (and k(cat)/K-m(arg)) among AKs, and a lack of synergistic substrate binding (K-d/K-m similar to 1). (c) 2007 Elsevier Ltd. All rights reserved.