Site-directed mutagenesis at the regulatory site of fructose 6-phosphate-1-kinase from Bacillus stearothermophilus

1988 ◽  
Vol 156 (1) ◽  
pp. 537-542 ◽  
Author(s):  
B.C. Valdez ◽  
S.H. Chang ◽  
E.S. Younathan
Keyword(s):  
Bacillus Stearothermophilus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Regulatory Site

Site-directed mutagenesis of Bacillus stearothermophilus lactate dehydrogenase

1987 ◽  
Vol 15 (1) ◽  
pp. 152-153 ◽  
Author(s):  
ANTHONY R. CLARKE ◽  
DALE B. WIGLEY ◽  
DAVID A. BARSTOW ◽  
WILLIAM N. CHIA ◽  
ADAM D. B. WALDMAN ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Site-Directed Mutagenesis of a Thermostable α-Amylase from Bacillus stearothermophilus: Putative Role of Three Conserved Residues1

1990 ◽  
Vol 107 (2) ◽  
pp. 267-272 ◽  
Author(s):  
Mauno Vihinen ◽  
Pauli Olikka ◽  
Jukka Niskanen ◽  
Peter Meyer ◽  
IIari Suominen ◽  
...  
Keyword(s):  
Bacillus Stearothermophilus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Putative Role

scholarly journals Increasing the thermostability of the neutral proteinase of Bacillus stearothermophilus by improvement of internal hydrogen-bonding

1992 ◽  
Vol 285 (2) ◽  
pp. 625-628 ◽  
Author(s):  
V G Eijsink ◽  
G Vriend ◽  
J R Van der Zee ◽  
B Van den Burg ◽  
G Venema
Keyword(s):  
Molecular Dynamics ◽  
Hydrogen Bond ◽  
Bacillus Stearothermophilus ◽  
Site Directed Mutagenesis ◽  
Internal Cavity ◽  
Directed Mutagenesis ◽  
Neutral Proteinase ◽  
Internal Hydrogen ◽  
Internal Hydrogen Bond ◽  
Dynamics Simulations

In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine. Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond. In addition, the hydroxy group of Ser-170 could contribute to stability by filling an internal cavity. After the introduction of the mutation, using site-directed-mutagenesis techniques, an increase in stability of 0.7 +/- 0.1 degrees C was obtained.

scholarly journals Probing the Essential Catalytic Residues and Substrate Affinity in the Thermoactive Bacillus stearothermophilus US100 l-Arabinose Isomerase by Site-Directed Mutagenesis

2007 ◽  
Vol 189 (9) ◽  
pp. 3556-3563 ◽  
Author(s):  
Moez Rhimi ◽  
Michel Juy ◽  
Nushin Aghajari ◽  
Richard Haser ◽  
Samir Bejar
Keyword(s):  
Amino Acids ◽  
Bacillus Stearothermophilus ◽  
Catalytic Mechanism ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Isomerization Reaction ◽  
Directed Mutagenesis ◽  
Substrate Affinity ◽  
Sequence Alignments ◽  
Arabinose Isomerase

ABSTRACT The l-arabinose isomerase (l-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of l-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 l-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 l-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the l-fucose isomerase and the l-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 l-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 l-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for l-fucose and decreased the affinity for l-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.

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