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 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 High-Throughput Multiplex Flow Cytometry Screening for Botulinum Neurotoxin Type A Light Chain Protease Inhibitors

2010 ◽  
Vol 8 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Matthew J. Saunders ◽  
Steven W. Graves ◽  
Larry A. Sklar ◽  
Tudor I. Oprea ◽  
Bruce S. Edwards
Keyword(s):  
Flow Cytometry ◽  
Protease Inhibitors ◽  
High Throughput ◽  
Light Chain ◽  
Botulinum Neurotoxin ◽  
Type A ◽  
Botulinum Neurotoxin Type A

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.

scholarly journals Regulated Secretion Is Impaired in AtT-20 Endocrine Cells Stably Transfected with Botulinum Neurotoxin Type A Light Chain

1997 ◽  
Vol 272 (41) ◽  
pp. 26005-26008 ◽  
Author(s):  
Fernando Aguado ◽  
Lourdes Gombau ◽  
Glòria Majó ◽  
Jordi Marsal ◽  
Jerónimo Blanco ◽  
...  
Keyword(s):  
Light Chain ◽  
Botulinum Neurotoxin ◽  
Endocrine Cells ◽  
Type A ◽  
Regulated Secretion ◽  
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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