Engineering of halophilic enzymes: two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases.
Nucleoside diphosphate kinase from Halomonas sp. 593 (HaNDK) exhibits halophilic characteristics. Residues 134 and 135 in the carboxy-terminal region of HaNDK are Glu-Glu, while those of its homologous counterpart of non-halophilic Pseudomonas NDK (PaNDK) are Ala-Ala. The double mutation, E134A-E135A, in HaNDK results in the loss of the halophilic characteristics, and, conversely, the double mutation of A134E-A135E in PaNDK confers halophilic characters to this enzyme, indicating that the charged state of these two residues that are located in the C-terminal region plays a critical role in determining halophilic characteristics. The importance of these two residues versus the net negative charges will be discussed in relation to the halophilicity of NDK.
Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan.
Hiroko
H
Tokunaga
Tsutomu
T
Arakawa
Masao
M
Tokunaga
D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D020632:Halomonas / Q000201:enzymology*; D006358:Hot Temperature; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D011549:Pseudomonas / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013997:Time Factors
D024342:Amino Acids, Acidic; D012965:Sodium Chloride; D009701:Nucleoside-Diphosphate Kinase