Biophysical Investigation of the Function of Enzyme 3DL1
Biophysical Investigation of the Function of Enzyme 3DL1
复制标题
酶 3DL1 功能的生物物理研究
DOI：
10.1016/j.bpj.2019.11.2914
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发表时间：
2020
影响因子：
3.4
通讯作者：
Koeppe, Julia R.
中科院分区：
文献类型：
--
作者：
Khan, Ali A.;Kirsch, Michael;Koeppe, Julia R.
蛋白质的功能通常与结构有关，因此对功能未知的蛋白质或酶进行结构比较可能有助于阐明其功能。从克雷伯菌肺炎中提取的一种功能未知的酶（PDB ID 3DL1）已经用各种方法来预测和测试酶的功能。有和没有锌结合的3DL1的晶体结构表明，锌结合的位点与已知金属肽酶的序列和结构相似。这些晶体结构也表明锌结合影响了蛋白质的二级结构。将3DL1与其他蛋白质序列和结构进行序列和结构比较的计算方法。这表明3DL1可能是一种具有肽酶活性的水解酶。3DL1在大肠杆菌细胞中表达，并通过金属亲和层析纯化。采用隔色层析进一步纯化3DL1。利用圆二色性（CD）光谱研究了3DL1在有锌和无锌情况下的二级结构。CD数据允许测定锌离子与蛋白质结合的化学计量学。预计每个蛋白质分子会结合一个锌离子。此外，使用各种相关底物进行酶促测定，以监测存在和不存在锌的3DL1的活性。这些测定是用小分子底物进行的，并通过紫外/可见光谱进行监测。由于3DL1在结构上与明胶酶A有一定的相似性，因此在明胶实验中也测试了具有较大蛋白底物的3DL1的活性。这个项目的相关性来自于鉴定未知功能蛋白质的潜在酶促作用的必要性。通过这种方法，一些蛋白质可能对治疗人类疾病有用，或者有助于更好地了解疾病的原因。
Protein function is often tied to structure, so structural comparisons for a protein or enzyme of unknown function may help to elucidate its function. An enzyme of unknown function (PDB ID 3DL1) derived from Klebsiella pneumonia has been studied using various methods to predict and test enzyme function. Crystal structures of 3DL1 with and without zinc bound show that zinc binds at a site with sequence and structural similarity to known metallopeptidases. These crystal structures also show that zinc binding affects the secondary structure of the protein. Computational methods for sequence and structure comparisons were made of 3DL1 to other protein sequences and structures. These show that 3DL1 may be a hydrolase with possible peptidase activity. 3DL1 was expressed in E. coli cells and purified via metal-affinity chromatography. Size exclusion chromatography was used to further purify 3DL1. To study the secondary structure of 3DL1 in the presence and absence of zinc, circular dichroism (CD) spectroscopy was used. The CD data allowed for determination of the stoichiometry of binding of the zinc ion (s) to the protein. It was expected that one zinc ion would bind per protein molecule. Moreover, enzymatic assays were performed using various relevant substrates to monitor the activity of 3DL1 in the presence and absence of zinc. These assays were performed with small molecule substrates and were monitored by UV/visible spectroscopy. Because 3DL1 has some structural similarity to gelatinase A, the activity of 3DL1 with a larger protein substrate was also tested in a gelatin-based assay. The relevance of this project comes from the necessity to identify potential enzymatic action of proteins of unknown function. Through this, some proteins could become useful for treatment of human disease or lead to a greater understanding of their cause.