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 r 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 ATPindependent manner. In thermal denaturation experiments, Bt-Lon was found to display an indicator of thermostability value, T m 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.

Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

“…The critical requirement for ATP in our Lon-1 chaperone assay results is in agreement with that reported for chaperones in general [64] and for yeast Lon [58],[60], but differs with one study in which the chaperone-like activity of a bacterial Lon was investigated. In that report, Lon from Brevibacillus thermoruber prevented insulin aggregation in an ATP-independent manner [59].…”

“…In addition to its function as an ATP-dependent protease, Lon exhibits a chaperone-like role in mammalian mitochondria, yeast, and bacteria [58],[59],[60],[61],[62]. Cloud et al [28] reported that complementation of an E. coli lpxA temperature-sensitive mutant with sequence corresponding to the N-terminus and ATP-binding site of B. burgdorferi lon-1 allowed the mutant to grow at the non-permissive temperature.…”

“…The chaperone assay is based upon the prevention of B-chain aggregation through interaction with an added chaperone. Assay results are measured via light scattering caused by insulin aggregates [59],[61]. Therefore, to determine if rLon-1 possessed chaperone-like activity, we tested the ability of rLon1 and rLon-1 S714A to inhibit the aggregation of insulin B-chain under reducing conditions.…”

Role of the Inserted α-Helical Domain in E. coli ATP-Dependent Lon Protease Function

“…The formation of enzyme-DNA complexes was monitored by a deceleration of DNA in an agarose gel (GMSA method) [21]. 20–25 μg of Lon-H 6 or Lon-dHI(CC) were incubated for 30 min at 25°C with 500 ng of plasmid DNA (pET28a) in 25 μL of 20 mM Tris-HCl buffer, pH 7.5, containing 60 mM NaCl.…”

Optimization of Culture Conditions to Produce Thermostable Keratinolytic Protease of Brevibacillus thermoruber LII, Isolated from the Padang Cermin Hot Spring, Lampung, Indonesia

“…Wang et al (2012) reported about a Brevibacillus species that produced thermostable protease with slightly different characters to the one produced by strain LII (optimum temperature 75 °C and optimum pH 9). Another experiment on this bacteria was reported by Lee et al (2004). Lee's group reported the presence of a its ability to degrade fibroin which is one of the substrates of protease (Suzuki et al 2009).…”