Alkaline phosphatase from the Antarctic strain TAB5. Properties and psychrophilic adaptations.
The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TAB5 was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified. The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escerichia coli AP D153H, a homology-based 3D model of the TAB5 AP was constructed on the basis of which various features of the enzyme amino-acid sequence can be interpreted in terms of potential psychrophilic adaptations. The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TAB5 AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity. The use of this heat-labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.
Rina
M
M
Institute of Molecular Biology, Enzyme Technology Division, Heraklion, Crete, Greece.
Pozidis
C
C
Mavromatis
K
K
Tzanodaskalaki
M
M
Kokkinidis
M
M
Bouriotis
V
V
D000222:Adaptation, Physiological; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003080:Cold Temperature; D017124:Conserved Sequence / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics
D011994:Recombinant Proteins; D004247:DNA; D000469:Alkaline Phosphatase