Novel trimeric adenylate kinase from an extremely thermoacidophilic archaeon, Sulfolobus solfataricus: molecular cloning, nucleotide sequencing, expression in Escherichia coli, and characterization of the recombinant enzyme.
A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.
Okajima
Toshihide
T
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan. tokajima@sanken.osaka-u.ac.jp
Kitaguchi
Daisuke
D
Fujii
Kumiko
K
Matsuoka
Hidetada
H
Goto
Sachio
S
Uchiyama
Susumu
S
Kobayashi
Yuji
Y
Tanizawa
Katsuyuki
K
D000263:Adenylate Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016944:Sulfolobus / Q000201:enzymology*
D011994:Recombinant Proteins; D000263:Adenylate Kinase; D004259:DNA-Directed DNA Polymerase