Mutation study of conserved amino acid residues of Spirulina Δ6-acyl-lipid desaturase showing involvement of histidine 313 in the regioselectivity of the enzyme
Mutation study of conserved amino acid residues of Spirulina Δ6-acyl-lipid desaturase showing involvement of histidine 313 in the regioselectivity of the enzyme
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DOI：
10.1007/s00253-004-1655-x
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发表时间：
2004-11-01
影响因子：
5
通讯作者：
Cheevadhanarak, S
中科院分区：
文献类型：
--
作者：
Hongsthong, A;Subudhi, S;Cheevadhanarak, S
在蓝藻钝顶螺旋藻中，去饱和过程由三种去饱和酶进行：Delta（9）、Delta（12）和Delta（6）去饱和酶，分别由desC、德萨和desD编码。Δ（6）去饱和酶负责催化亚油酸，产生γ-亚麻酸（18：3（Δ 9，12，6）），该过程的最终产物。本研究利用pTrcHisA表达系统在大肠杆菌中表达desD基因。为了鉴定参与酶活性的氨基酸残基，使用各种生物体进行序列比较。比对结果显示，三个保守的组氨酸簇，一些保守的残基之间的所有列出的生物和一些保守的残基之间的蓝藻物种可能参与去饱和活性。在desD基因中产生一系列定点突变以评估这些残基相对维斯酶功能的作用。这一做法表明：（1）H313参与酶的区域选择性，（2）三个组氨酸簇与H313、H315、D138和E140一起是酶活性所需的，最有可能作为催化Fe中心的提供者，（3）W294也是Delta（6）去饱和酶活性所必需的，可能通过形成底物结合口袋的一部分。
In the cyanobacterium Spirulina platensis, the desaturation process is carried out by three desaturases: the Delta(9), Delta(12) and Delta(6) desaturases, encoded by desC, desA and desD, respectively. The Delta(6) desaturase is responsible for the catalysis of linoleic acid, yielding gamma-linolenic acid (18:3(Delta9,12,6)), the end-product of the process. In this study, the desD gene was expressed in Escherichia coli using a pTrcHisA expression system. In order to identify the amino acid residues involved in the enzymatic activity, a sequence comparison was performed using various organisms. The alignment revealed three conserved histidine clusters, a number of conserved residues among all listed organisms and a few conserved residues among cyanobacterial species possibly involved in the desaturation activity. A series of site-directed mutations were generated in the desD gene to evaluate the role of these residues vis-a-vis the enzyme function. This approach revealed that: (1) H313 is involved in the regioselectivity of the enzyme, (2) the three histidine clusters together with H313, H315, D138 and E140 are required for enzymatic activity, most likely as providers of the catalytic Fe center and (3) W294 is also essential for the activity of Delta(6) desaturase, possibly by forming part of the substrate-binding pocket.