Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR-249 and biochemical characterization of its thermostable recombinant enzyme.
A gene encoding thermostable Lon protease from Brevibacillus thermoruber WR-249 was cloned and characterized. The Br. thermoruber Lon gene (Bt-lon) encodes an 88 kDa protein characterized by an N-terminal domain, a central ATPase domain which includes an SSD (sensor- and substrate-discrimination) domain, and a C-terminal protease domain. The Bt-lon is a heat-inducible gene and may be controlled under a putative Bacillus subtilis sigmaA-dependent promoter, but in the absence of CIRCE (controlling inverted repeat of chaperone expression). Bt-lon was expressed in Escherichia coli, and its protein product was purified. The native recombinant Br. thermoruber Lon protease (Bt-Lon) displayed a hexameric structure. The optimal temperature of ATPase activity for Bt-Lon was 70 degrees C, and the optimal temperature of peptidase and DNA-binding activities was 50 degrees C. This implies that the functions of Lon protease in thermophilic bacteria may be switched, depending on temperature, to regulate their physiological needs. The peptidase activity of Bt-Lon increases substantially in the presence of ATP. Furthermore, the substrate specificity of Bt-Lon is different from that of E. coli Lon in using fluorogenic peptides as substrates. Notably, the Bt-Lon protein shows chaperone-like activity by preventing aggregation of denatured insulin B-chain in a dose-dependent and ATP-independent manner. In thermal denaturation experiments, Bt-Lon was found to display an indicator of thermostability value, Tm of 71.5 degrees C. Sequence comparison with mesophilic Lon proteases shows differences in the rigidity, electrostatic interactions, and hydrogen bonding of Bt-Lon relevant to thermostability.
Lee
Alan Y-L
AY
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Tsay
San-San
SS
Chen
Mao-Yen
MY
Wu
Shih-Hsiung
SH
D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D049070:Protease La; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La