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Eur J Biochem. 2004 Feb;271(4):834-44.

Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR-249 and biochemical characterization of its thermostable recombinant enzyme.

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Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.


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.

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