[2S06a] 23.Protein and Metabolic Engineering

2:05 PM - 2:45 PM JST(5:05 AM - 5:45 AM UTC)

[2PME-02] Structural and Functional Studies of Proline-Specific Peptidases from Lactococcus lactis: Insights into Enzyme Regulation, Substrate Discrimination, and Engineering

○Takuji Tanaka¹, Shangyi Xu¹ (1. University of Saskatchewan (Canada))

Keywords:

food fermentation,bitter taste,protein engineering,direct evolution,allosteric enzyme

Proline-specific peptidases are a unique group of enzymes involved in the hydrolysis of proline-containing small peptides, which resist to other peptidases due to the structure and conformational constraints of proline. These enzymes in Lactococcus lactis are of particular interest because they assist in the breakdown of these bitter peptides accumulated during food fermentation. We investigated the structure–function relationships of two homologous proline-specific peptidases from L. lactis, prolidase and aminopeptidase P (APP), with the long-term goal of engineering improved biocatalysts for food applications.Prolidase, a strict Xaa–Pro dipeptidase, displays allosteric behaviour and substrate inhibition, making it less desirable for industrial applications. X-ray crystallographic analysis revealed that prolidase was a homodimer with Loop 36-40 in the subunit interface. Mutations at residues in the interface (e.g., Asp36, His38, and Arg293) produced some variants showing no allosteric behaviour and/or substrate inhibition, highlighting the loop’s role in mediating its unique characteristics through inter-subunit interactions. The results indicated that the allostery of prolidase is caused by dimer arrangements in which the two monomers are positioned closer, whereas the non-allosteric mutants show increased inter-subunit distances and a reduced interface area. These findings support a structural model in which conformational features across the dimer interface regulate substrate access and dictate kinetic properties.While prolidase exclusively hydrolyzes dipeptides, APP cuts longer peptides where the second amino acid from the N-terminal is proline. We solved the structure of APP at 2.35 Å resolution. Prolidase and APP share a high degree of homology, with an r.m.s.d. of 0.78 Å between 204 pruned atom pairs. This comparison suggested that prolidase specifies its activity to dipeptides through Arg40, which forms an electrostatic interaction with the dipeptide carboxyl terminus during substrate binding and sterically hinders the entry of longer peptides. APP does not have equivalent structure element of Loop 36–40 from prolidase, and this structural difference allows the accommodation of extended peptides. Based on this observation, we attempted to combine the properties of the two enzymes into a single enzyme to broaden substrate specificity, i.e., to have prolidase hydrolyze longer peptides, by modifying the substrate-binding region and dimer interface of prolidase. Some engineered prolidase variants, such as Δ101, and Δ101/R293S, showed activities toward tripeptide substrates while retaining dipeptidase activity. Moreover, domain-swap studies confirmed that the N-terminal domains of APP and prolidase are key determinants of substrate discrimination. Overall, this work provides a structural basis for substrate discrimination in proline-specific peptidases, supporting efforts to engineer enhanced enzymes for food applications.