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

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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).