Probing the Roles of Active Site Residues in Phosphatidylinositol-Specific Phospholipase C fromBacillus cereusby Site-Directed Mutagenesis†

Biochemistry ◽  
1997 ◽  
Vol 36 (42) ◽  
pp. 12802-12813 ◽  
Author(s):  
Claudia S. Gässler ◽  
Margret Ryan ◽  
Tun Liu ◽  
O. Hayes Griffith ◽  
Dirk W. Heinz
Keyword(s):  
Phospholipase C ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues

scholarly journals Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds

Glycobiology ◽  
1998 ◽  
Vol 8 (10) ◽  
pp. 1021-1028 ◽  
Author(s):  
G. Garcia-Casado ◽  
C. Collada ◽  
I. Allona ◽  
R. Casado ◽  
L. F. Pacios ◽  
...  
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Class I ◽  
Directed Mutagenesis ◽  
Active Site Residues

scholarly journals Heterologous Expression of the Vanadium-containing Chloroperoxidase fromCurvularia inaequalisinSaccharomyces cerevisiaeand Site-directed Mutagenesis of the Active Site Residues His496, Lys353, Arg360, and Arg490

1999 ◽  
Vol 274 (34) ◽  
pp. 23820-23827 ◽  
Author(s):  
Wieger Hemrika ◽  
Rokus Renirie ◽  
Sandra Macedo-Ribeiro ◽  
Albrecht Messerschmidt ◽  
Ron Wever
Keyword(s):  
Heterologous Expression ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues

Site-directed mutagenesis of active site residues in Bacillus subtilis α-amylase

1992 ◽  
Vol 1120 (3) ◽  
pp. 281-288 ◽  
Author(s):  
Kenji Takase ◽  
Takashi Matsumoto ◽  
Hiroshi Mizuno ◽  
Kazio Yamane
Keyword(s):  
Bacillus Subtilis ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A

2001 ◽  
Vol 288 (5) ◽  
pp. 1231-1237 ◽  
Author(s):  
Michela Rigoni ◽  
Paola Caccin ◽  
Eric A. Johnson ◽  
Cesare Montecucco ◽  
Ornella Rossetto
Keyword(s):  
Light Chain ◽  
Active Site ◽  
Botulinum Neurotoxin ◽  
Type A ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Botulinum Neurotoxin Type A

scholarly journals Identification of core active site residues of the sulfur oxygenase reductase fromAcidianus ambivalensby site-directed mutagenesis

2005 ◽  
Vol 248 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Tim Urich ◽  
Anja Kroke ◽  
Christian Bauer ◽  
Kerstin Seyfarth ◽  
Muriel Reuff ◽  
...  
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Sulfur Oxygenase Reductase
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