DnaK from Vibrio proteolyticus: complementation of a dnaK-null mutant of Escherichia coli and the role of its ATPase domain.
We cloned the 4.8-kbp DNA fragment containing the dnaK gene from the chromosomal DNA of Vibrio proteolyticus. It contained four genes arranged unidirectionally in the order of grpE, gltP, dnaK and dnaJ. The DnaK gene of V. proteolyticus (VprDnaK) allowed a dnaK-null mutant of Escherichia coli (DeltadnaK52) to propagate lambda phages but not to grow at 43 degrees C. However, a chimeric DnaK gene comprising the regions corresponding to the N-terminal ATPase domain of E. coli DnaK (EcoDnaK) and the C-terminal region of VprDnaK including the substrate-binding domain, enabled the mutant to grow at 43 degrees C. The temperature dependence for the ATPase activity of the chimeric DnaK was similar to that of EcoDnaK. Fluorometric analyses showed that the chimeric DnaK is much more thermostable than EcoDnaK and VprDnaK. These findings indicate that the thermal stability of the ATPase domain of DnaK is responsible for its chaperone action at high temperatures such as 43 degrees C.
Yoshimune
Kazuaki
K
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Galkin
Andrey
A
Kulakova
Ljudmila
L
Yoshimura
Tohru
T
Esaki
Nobuyoshi
N
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013696:Temperature; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli