Cloning, analysis, and expression of the gene for inorganic pyrophosphatase of Aquifex pyrophilus and properties of the enzyme.
The gene encoding Aquifex pyrophilus (Apy) pyrophosphatase was cloned and sequenced. The deduced amino acid sequence of Apy pyrophosphatase showed a 94.2% homology to Aquifex aeolicus (Aae) pyrophosphatase. The gene exhibits a difference in the codon usage at the third position from Aae pyrophosphatase. The gene was expressed under the control of a tac promoter in E. coli. The recombinant Apy pyrophosphatase was purified 18.7-fold with a 52.8% yield and a specific activity of 26.2 U mg(-1) protein. The native enzyme has a homotetramer of 177 amino acids. The enzyme shows optimal activity in pH 7.5. The optimum temperature was approximately 70 degrees C. A divalent cation was absolutely required for the enzyme activity; Mg2+ was the most effective.
Hoe
Hyang-Sook
HS
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Korea.
Kim
Hyun-Kyu
HK
Kwon
Suk-Tae
ST
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
D011755:Pyrophosphatases