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。在二十种常见的氨基酸中，由于其结构与其他氨基酸的不同，它是非常独特的。这种差异导致了Pro对蛋白质水解的抵抗力，这导致了发酵食品特有的苦味(含Pro的多肽是苦味的)，并在蛋白质水解中需要Pro专一性多肽酶(在胶原蛋白中循环Pro和消化食物中的蛋白质)。脯氨酸酶是一种重要的脯氨酸专一性多肽酶，是唯一能降解X-Pro的多肽酶。这项拟议的研究将通过1)确定酶的三维结构，以及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)，来研究其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Prolidase将用五种不同的策略结晶。获得的晶体将在加拿大光源公司进行X射线衍射数据收集。通过对衍射数据的分析，建立了丙氨酸酯酶的三维模型。在模型的基础上，将确定负责独特功能的残基，并将使用每个残基的突变脯氨酸酶来确认它们的作用。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).