Primary structure, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothermophilus.
The complete primary structure of inorganic pyrophosphatase [EC 3.6. 1.1] from Bacillus stearothermophilus (ATCC 12016) was determined at the amino acid level by automated Edman degradation. The subunit of the enzyme consists of 164 amino acid residues with a calculated molecular mass of 18,796. The amino acid sequence of the enzyme is almost identical to that of thermophilic bacterium PS-3. Based on the determined primary structure, a PCR-amplified semi-synthetic gene was constructed and expressed in Escherichia coli JM109. The recombinant Bst. PPase showed the same characteristics and activity as the authentic enzyme, and exhibits higher thermostability than the E. coli enzyme. Furthermore, we prepared tyrosine-substituted variants by site-directed mutagenesis to elucidate the role of two highly conserved tyrosines (Y46 and Y130). As a result, two variants, Y46F and Y130F, lost most of their enzyme activity, whereas their conformations were unaffected. However, the wild-type and two variants exhibited different thermostability behaviors in the presence or absence of Mg2+. Therefore, these tyrosines may contribute to the structural integrity of the active site of the enzyme.
Satoh
T
T
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Chitosedai, Setagaya-ku, Tokyo, 157-8572, Japan.
Shinoda
H
H
Ishii
K
K
Koyama
M
M
Sakurai
N
N
Kaji
H
H
Hachimori
A
A
Irie
M
M
Samejima
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002942:Circular Dichroism; D017124:Conserved Sequence; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D043564:Inorganic Pyrophosphatase; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014443:Tyrosine
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase