Molecular mechanism of distinct salt-dependent enzyme activity of two halophilic nucleoside diphosphate kinases.
Nucleoside diphosphate kinases from haloarchaea Haloarcula quadrata (NDK-q) and H. sinaiiensis (NDK-s) are identical except for one out of 154 residues, i.e., Arg(31) in NDK-q and Cys(31) in NDK-s. However, the salt-dependent activity profiles of NDK-q and NDK-s are quite different: the optimal NaCl concentrations of NDK-q and NDK-s are 1 M and 2 M, respectively. We analyzed the relationships of the secondary, tertiary, and quaternary structures and NDK activity of these NDKs at various salt concentrations, and revealed that 1), NDK-q is present as a hexamer under a wide range of salt concentrations (0.2-4 M NaCl), whereas NDK-s is present as a hexamer at an NaCl concentration above 2 M and as a dimer at NaCl concentrations below 1 M; 2), dimeric NDK-s has lower activity than hexameric NDK-s; and 3), dimeric NDK-s has higher helicity than hexameric NDK-s. We also determined the crystal structure of hexameric NDK-q, and revealed that Arg(31) plays an important role in stabilizing the hexamer. Thus the substitution of Arg (as in NDK-q) to Cys (as in NDK-s) at position 31 destabilizes the hexameric assembly, and causes dissociation to less active dimers at low salt concentrations.
Yamamura
Akihiro
A
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Ichimura
Takefumi
T
Kamekura
Masahiro
M
Mizuki
Toru
T
Usami
Ron
R
Makino
Tsukasa
T
Ohtsuka
Jun
J
Miyazono
Ken-ichi
K
Okai
Masahiko
M
Nagata
Koji
K
Tanokura
Masaru
M
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003460:Crystallization; D004926:Escherichia coli; D019613:Haloarcula; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000235:genetics; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012965:Sodium Chloride / Q000737:chemistry*; D040681:Structural Homology, Protein; D014961:X-Ray Diffraction
D019843:Archaeal Proteins; D012965:Sodium Chloride; D009701:Nucleoside-Diphosphate Kinase