Pk-cdcA encodes a CDC48/VCP homolog in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1: transcriptional and enzymatic characterization.
A gene encoding a cell division control protein from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, Pk-cdcA, was cloned and sequenced. The Pk-cdcA gene is composed of 2508 nucleotides, encoding a protein of 835 amino acids with a molecular mass of 93,666 Da. Pk-CdcA has a typical Walker-type ATPase motif and was classified as a new member of the CDC48/VCP subfamily of so-called AAA proteins. In addition, Pk-CdcA possesses a unique region composed of charged amino acids, which is not observed in other homologs from Archaea. Transcription of the gene was analyzed by primer extension and Northern analyses, revealing that Pk-cdcA is transcribed from a site 77 bases upstream of the initiation codon. Pk-CdcA and its deletion mutant Pk-CdcAdelta63, which lacks the unique inserted region, were expressed in Escherichia coli cells as His-tagged fusion proteins and purified. Both Pk-CdcA and Pk-CdcAdelta63 possess an ATPase activity, as do other CDC48/VCP proteins. However, Pk-CdcAdelta63 showed a higher level of ATPase activity and greater thermostability than Pk-CdcA. Furthermore, Pk-CdcAdelta63 has a higher Vmax value than wild type, even though the Km was unchanged. These observations indicated that the inserted region affects enzyme stability and activity. In order to investigate intracellular expression levels of Pk-CdcA, Western analysis was performed using anti-Pk-CdcA antisera obtained from immunized BALB/C mice. Equal levels of Pk-CdcA expression were observed during exponential and stationary phases. Growth phase-specific fragmentation of Pk-CdcA was found in stationary-phase cells.
Jeon
S J
SJ
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
Fujiwara
S
S
Takagi
M
M
Imanaka
T
T
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics*; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D019641:DNA, Archaeal; D015870:Gene Expression; D019766:Genes, Archaeal; D008969:Molecular Sequence Data; D019711:Pyrococcus / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D000074405:Valosin Containing Protein
D019843:Archaeal Proteins; D018797:Cell Cycle Proteins; D019641:DNA, Archaeal; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C402119:cdcA protein, Pyrococcus kodakaraensis; D000074405:Valosin Containing Protein; C000614179:Vcp protein, mouse