A Cys/Ser mutation of NDPK-A stabilizes its oligomerization state and enhances its activity.
Nucleoside diphosphate phosphate transferase A (NDPK-A) has been shown to play critical roles in the regulation of proliferation, differentiation, growth and apoptosis of cells. Our previous study suggested that the disulphide cross-linkage between cysteine 4 (C4) and cysteine 145 (C145) of NDPK-A might be a possible regulator of its activity. To confirm this hypothesis, the C145 residue of NDPK-A was mutated to serine, and the isomerization and biological activities of the mutant were investigated and compared with those of its wild-type counterpart. It was found the C145S mutation eliminated the intramolecular disulphide bond (DB) and prevented the formation of intermolecular DB, which was known to dissociate the hexameric NDPK-A into dimeric one. We also demonstrated that the C145S mutation didn't affect the autologous hexamerization of this protein, and the mutant had increased bioactivities including phosphate transferase and DNase. These findings support the hypothesis that the formation of DBs in NDPK-A is involved in the regulation of the oligomerization and bioactivity of this multiple function protein, and that C145 is a key residue in the regulation of NDPK-A. In addition, the C145S mutant that we have constructed might be an attractive candidate for use in applications that require NDPK-A.
Chen
Yunru
Y
Biomedical R&D Center, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, Guangdong, P. R. China 510632.
Qian
Chuiwen
C
Guo
Chaowan
C
Ge
Feng
F
Zhang
Xiaowei
X
Gao
Xue
X
Shen
Shan
S
Lian
Bing
B
Kitazato
Kaio
K
Wang
Yifei
Y
Xiong
Sheng
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003851:Deoxyribonucleases / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D054778:NM23 Nucleoside Diphosphate Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055503:Protein Multimerization / Q000235:genetics; D012694:Serine / Q000235:genetics
D004220:Disulfides; D054778:NM23 Nucleoside Diphosphate Kinases; D012694:Serine; D003851:Deoxyribonucleases; D003545:Cysteine