Homology modelling of cell-envelope proteinase

Engineering of the substrate binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactisProtein Engineering 6 (1993) 927-937

Roland Siezen, Paul Bruinenberg, Pieter Vos, Ingrid van Alen-Boerrigter, Monique Nijhuis, Arno Alting, Fred Exterkate, and Willem de Vos

Flavour formation in cheese

MilkMilk

Lactose/Lactose/ citric acidcitric acid

CaseinCasein FatFat

peptidespeptides

amino acidsamino acids

flavour compoundsflavour compounds

fatty acidsfatty acids

chymosinprotease

peptidases

amino acidconvertases

lipasesesterases

metabolic enzymes

pyruvatepyruvate

metabolic enzymes

Proteolysis by lactic acid bacteria

L.lactis

Casein peptides

PrtP PepN

PepXPepA

N

C

Ca1

Ca2

Ser

His Asp

+28

+8

+14

+15

substratebinding cleft

PrtP protease domain

+151

I-domain

137-139

166

s1-casein(1-23) degradation by PrtP

R-P-K-H-P-I-K-H-Q-G-L-P-Q-E-V-L-N-E-N-L-L-R-F

wild-type

N166D

AKT(137-139)GDT

AKT(137-139)GPP

AKT(137-139)GLA

238-388)

5 10 15 20

Main cleavage sites: pH 6.5, low NaCl

Conclusions

Homology modelling• substrate-binding region of PrtP can be modelled• several (large) loops cannot be modeled• model suffices for general predictions

Protein engineering• mutations in substrate-binding region, based on homology modelling• mutants show altered proteolytic specificity• tested mutants under cheese making conditions