The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site.
A gene with significant similarity to bacterial Lon proteases was identified during the sequencing of the genome of the thermoacidophilic archaeon Thermoplasma acidophilum. Protein sequence comparison revealed that Thermoplasma Lon protease (TaLon) is more similar to the LonB proteases restricted to Gram-positive bacteria than to the widely distributed bacterial LonA. However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases. Using site-directed mutagenesis we show here that TaLon and EcLon, and probably all other Lon proteases, contain a Ser-Lys dyad active site. The TaLon active site mutants were fully assembled and, similar to TaLon wild-type, displayed an apparent molar mass of 430 kDa upon gelfiltration. This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown symmetry. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective protease active site mutants revealed differences in Km and V values. This suggests that in the course of protein degradation by wild-type Lon the protease domain might influence the activity of the ATPase domain.
Besche
Henrike
H
Department of Structural Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany.
Zwickl
Peter
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D049070:Protease La / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013822:Thermoplasma / Q000201:enzymology*
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D012694:Serine; D049070:Protease La; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine