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Structure-function relationship studies on unique allosteric prolidase, which may lead value-added foods, using X-ray cristallography and protein engineering techniques

利用 X 射线晶体学和蛋白质工程技术研究独特的变构脯氨酸酶的结构-功能关系，该酶可能导致食品增值

基本信息

批准号：
283277-2010
负责人：
Tanaka, Takuji
金额：
$ 1.97万
依托单位：
University of Saskatchewan
依托单位国家：
加拿大
项目类别：
Discovery Grants Program - Individual
财政年份：
2014
资助国家：
加拿大
起止时间：
2014-01-01 至 2015-12-31
项目状态：
已结题

来源：
https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=548100
关键词：
Structure function relationship studies unique

项目摘要

Proteins, polymers of amino acids, are essential biological substances and are the focus of research and applications in foods, agricultural products, and medicine. Combinations of amino acids determine the characteristics and functions of the proteins. Among the twenty common amino acids, proline is very unique because of its structural difference from other amino acids. This difference results in the resistance of proline to protein hydrolysis, which leads to the characteristic bitter flavour of fermented foods (proline-containing peptides are bitter), and to requirements for proline-specific peptidases in protein hydrolysis (to recycle proline in collagen and to digest proteins in foods). One important proline-specific peptidase is prolidase, which is the exclusive peptidase for hydrolyzing X-Pro. This proposed research will investigate how the structural characteristics of prolidase relate to its unique functions by 1) determining the three-dimensional structure, and 2) examining the functional factors (such as specific amino acid residues or specific local structures) of the enzyme using protein engineering techniques. Prolidase, provided using our recombinant system, will be crystallized with five different strategies. The obtained crystals will be subjected to the X-ray diffraction data collection at Canadian Light Source. The diffraction data will be analyzed to construct the three-dimensional models of prolidase. Based on the models, the residues responsible for the unique functions will be identified, and their roles will be confirmed using mutant prolidases for each residue. We expect that results from this research will ultimately contribute to food processing (such as debittering of fermented foods) and medical applications (such as treatment of prolidase deficiency).

蛋白质是氨基酸的聚合物，是必需的生物物质，是食品、农产品和医药领域研究和应用的焦点。氨基酸的组合决定了蛋白质的特性和功能。在20种常见的氨基酸中，脯氨酸因其结构与其他氨基酸不同而非常独特。这种差异导致脯氨酸对蛋白质水解的抗性，这导致发酵食品的特征性苦味（含脯氨酸的肽是苦的），以及在蛋白质水解中对脯氨酸特异性肽酶的需求（以回收胶原蛋白中的脯氨酸并消化食品中的蛋白质）。一种重要的脯氨酸特异性肽酶是氨酰基脯氨酸二肽酶，其是水解X-Pro的唯一肽酶。本研究将通过1）确定三维结构，2）使用蛋白质工程技术检查酶的功能因子（如特定氨基酸残基或特定局部结构），研究氨脯氨酸二肽酶的结构特征与其独特功能之间的关系。使用我们的重组系统提供的脯氨酰二肽酶将用五种不同的策略结晶。所得晶体将在Canadian Light Source进行X射线衍射数据收集。衍射数据将进行分析，以构建的三维模型的氨酰基脯氨酸二肽酶。基于这些模型，将鉴定负责独特功能的残基，并使用每个残基的突变型氨酰基脯氨酸二肽酶确认其作用。我们预计，这项研究的结果最终将有助于食品加工（如发酵食品的脱苦）和医疗应用（如氨脯氨酸二肽酶缺乏症的治疗）。