Characterization of a new AAA+ protein from archaea.
We investigated a new archaeal member of the AAA+ protein family (ATPases associated with various cellular activities) which is found in all methanogenic archaea and the sulphate-reducer Archaeoglobus fulgidus. These proteins cluster to COG1223 predicted to form a subgroup of the AAA+ ATPases. The gene from A. fulgidus codes for a protein of 40 kDa monomeric molecular weight, which we overexpressed in Escherichia coli and purified to homogeneity. The protein forms ring-shaped complexes with a diameter of 125A as determined by electron microscopy. Using sedimentation equilibrium analysis we demonstrate that it assembles into hexamers over a wide concentration range both in presence and absence of ATP. As suggested by homology to other members of the AAA+ family, the complex binds and hydrolyzes ATP. Michaelis-Menten analysis revealed a k(cat) of 118 min(-1) and a K(M) of 1.4 mM at 78 degrees C. This hyperthermophilic archaeal ATPase is stable to 86 degrees C and the ATPase activity is maximal at this temperature. The protein is most homologous to the AAA-domain of FtsH from bacteria, while the N-terminal domain shows predicted structural homology to members of the CDC48 family of AAA proteins. Possible roles of this new AAA+ protein are discussed.
Summer
Heike
H
Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland.
Bruderer
Roland
R
Weber-Ban
Eilika
E
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D019611:Archaeoglobus fulgidus / Q000201:enzymology / Q000235:genetics; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D019843:Archaeal Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases