Construction of super-stable enzymes : distinct reversibility of structure and its mechanism

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(1) We have succeeded in the expression of haloarchaeal nucleoside diphosphate in Escherichia coil and X-ray structure analysis. The structure of main chain of polypeptide is quite similar to non-halophilic counterpart, indicating elegant distribution of side chains of acidic amino acid residues.(2) We have cloned nucleoside diphosphate kinase gene from moderately halophilic bacteria and expressed it in E. coil. We found for the first time it forms dimeric structure.(3) We have succeeded in the construction, expression, purification and characterization of chimeric nucleoside diphosphate kinase molecules from the enzymes isolated from Halomonas and Pseudomonas, which is halophilic and non-halophilic bacteria, respectively.Consequently, the landmark of halophilic protein is found to be(1) higher optimum salt concentration for enzymatic activity(2) higher stability at higher salt environment(3) highly reversible characteristics due to its high solubility(4) slow mobility on SDS-polyacrylamide gel electrophoresis.