The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum
The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum
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DOI：
10.1016/j.mcat.2017.12.033
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发表时间：
2018-02-01
影响因子：
4.6
通讯作者：
Humble, Maria Svedendahl
中科院分区：
文献类型：
--
作者：
Chen, Shan;Berglund, Per;Humble, Maria Svedendahl
来自紫色色杆菌（Cv-ATA）的胺转氨酶是一种吡哆醛-5 '-磷酸（PLP）依赖性酶。这种酶的生物活性需要形成全同二聚体。然而，由于二聚体解离，Cv-ATA的操作稳定性低。在酶二聚体界面，两个磷酸基团结合杯（PGBC）的位置。每个杯协调PLP的磷酸基团的氢键起源于两个亚基。假设磷酸基团（PLP或游离无机磷酸盐）与PGBC的分子配位可以影响二聚体稳定性和酶活性。为了测试这一假设，通过各种生物物理技术探索磷酸盐（作为PLP中的官能团或作为游离无机阴离子）对Cv-ATA的稳定性和活性的影响。结果表明，Cv-ATA对PLP具有相对低的亲和力，这导致在酶纯化后过量的载脂蛋白二聚体酶。在补充有PLP的缓冲溶液中孵育载脂蛋白二聚体恢复了活性全二聚体。将PLP或无机磷酸盐添加到酶储存溶液中保护Cv-ATA免于化学和长期储存解折叠。与使用HEPES缓冲液相比，使用磷酸盐缓冲液导致全酶的更快失活。这些结果为如何提高PLP依赖性酶的稳定性开辟了新的前景。(C)2017爱思唯尔B. V.保留所有权利。
The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5'-phosphate (PLP) dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer. The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes. (C) 2017 Elsevier B.V. All rights reserved.