Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.
Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D001219:Aspartate Aminotransferases / Q000302:isolation & purification* / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D002845:Chromatography; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry
D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate