Proline is a unique amino acid among 20 common amino acids. It has a conjugation between its side chain and nitrogen, and the peptide bond to the flanking amino acid is an imino bond. This property makes peptide bonds involved with proline insensitive to general proteinases/peptidases, accumulating proline-containing peptides in food fermentation. The proline-containing peptides are generally bitter, and fermented foods often show the bitterness from them. Prolidase is a proline-specific dipeptidase that hydrolyzes Xaa-Pro dipeptides. It is considered as a useful enzyme to reduce the bitterness in fermented foods. <i>Lactococcus lactis</i> prolidase has shown a unique characteristics among prolidase, where allosteric behaviour and substrate inhibition are observed. Our group has investigated these properties and have made several mutant enzymes that do not show either or both of these unique characteristics. We have recently solved the X-ray structures of these mutant enzymes (H38S, D36S, and R293S) as well as wild-type enzyme at 2.3, 1.7, 2.7 and 2.3 Å, respectively. The models show that the active site is made of two histidine (His 204 and His 303) and Arg 40. A flexible loop structure from a monomer of this homodimeric enzyme interacts with the other monomer at His 303 and Arg 293 in and near the active site . This loop structure is an essential functional structure since a loop deletion has inactivated this enzyme. The models show the interaction of His38 on the loop to His303 in the active site and this interaction is one of the keys for allosteric behaviour which is governed by the interaction of two monomers. A slight shift of two monomers during the catalytic event is speculated to influence the allosteric behaviour. His38, Arg40 (both on the loop), Arg293, and His 303 (near or in the active site) form a pseudo substrate binding site, leading the substrate inhibition and allosteric behaviour. In this presentation, the details of these models will be discussed, and mechanisms of its unique characteristics and catalytic mechanism will be proposed.

X-ray crystallographic study of unique properties of <i>Lactococcus lactis</i> proline-specific dipeptidase, prolidase

ACS Fall 2021

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X-ray crystallographic study of unique properties of Lactococcus lactis proline-specific dipeptidase, prolidase