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

Abstract

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 σ A-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°C, and the optimal temperature of peptidase and DNA-binding activities was 50°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°C. Sequence comparison with mesophilic Lon proteases shows differences in the rigidity, electrostatic interactions, and hydrogen bonding of Bt-Lon relevant to thermostability.