Homology modelling of cell-envelope proteinase Engineering of the substrate binding region of the...
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Transcript of Homology modelling of cell-envelope proteinase Engineering of the substrate binding region of the...
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