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

蛋白质，氨基酸的聚合物，是必不可少的生物物质，是食品、农产品和医药研究和应用的重点。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的不同而显得十分独特。这种差异导致了脯氨酸对蛋白质水解的抵抗力，从而导致了发酵食品特有的苦味（含脯氨酸的肽是苦的），以及蛋白质水解对脯氨酸特异性肽酶的要求（在胶原蛋白中回收脯氨酸并消化食物中的蛋白质）。一个重要的脯氨酸特异性肽酶是脯氨酸酶，它是水解X-Pro的唯一肽酶。本研究将通过1)确定三维结构，2)利用蛋白质工程技术检测酶的功能因子（如特定的氨基酸残基或特定的局部结构）来研究脯氨酸酶的结构特征与其独特功能之间的关系。用我们的重组系统提供的增殖酶，将用五种不同的策略结晶。所得晶体将在加拿大光源进行x射线衍射数据采集。通过对衍射数据的分析，建立了蛋白酶的三维模型。基于这些模型，将识别出负责独特功能的残基，并使用每个残基的突变增殖酶来确认它们的作用。我们期望这项研究的结果最终将有助于食品加工（如发酵食品的脱臭）和医学应用（如治疗脯氨酸酶缺乏症）。

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).