Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme.
The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.
Izumi
M
M
Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan.
Fujiwara
S
S
Takagi
M
M
Kanaya
S
S
Imanaka
T
T
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D018833:Chaperonins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019711:Pyrococcus / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D000637:Transaminases / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; C121476:CpkA protein, archaeal; D019641:DNA, Archaeal; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000637:Transaminases; C071151:cobyric acid synthase; D000251:Adenosine Triphosphatases; D018833:Chaperonins