Mutational Analysis of a Surface Area that is Critical for the Thermal Stability of Thermolysin‐Like Proteases

Abstract

Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55 -69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp.59). In the case of the AspS7+Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.Keywords: thermal stability; thermolysin; autolysis ; calcium binding; unfolding pathway.Bacillus thermolysin-li ke proteases (TLP) are metallo-endopeptidases consisting of 300-3 19 residues. These enzymes contain one zinc ion that is essential for catalysis and they bind a varying number of calcium ions for which it is known that they contribute to stability [I -21. The amino acid sequences of many TLP are known and the crystal structures of thermolysin (the TLP of Bacillus thermoproteolyticus; [3,41) and TLP-cer (the TLP of Bacillus cereus; [S]) have been determined. TLP consist of an N-terminal domain (residues 1 -154; thermolysin numbering) characterized by a predominance of P-pleated sheet and a C-terminal domain (residues 155-31 6) that is mainly a-helical [4, 51. TLP differ in thermal stability [6] and the structural determinants of these differences have been analyzed by several sitedirected mutagenesis studies [7-111. At elevated temperatures TLP are irreversibly inactivated as a result of autolysis. Considering the broad specificity of TLP [ 12, 131, conformational Enzyme. Thermolysin (EC 3.4.24.27).proteolytic attack [14]. It has been shown that local unfolding processes that render the protein susceptible towards autolysis are the rate-limiting steps in thermal inactivation [6,[15][16]. It has been proposed that these thermally induced local unfolding processes mainly involve surface located regions of the protein 16, 9, 141. Accordingly, it has been shown that the changes in the stability of TLP-ste is most easily effected by mutation of surface-located residues that are clustered in one particular region (residues 55-69) of the protein [9, 111 (and see Figs 1 and 2). The difference in stability between thermolysin (tso = 86.5 "C ; see below for definition of tS0) and the TLP of B. stemothermophilus CU21 (TLP-ste; t50 = 73.4"C) ...

Gene Cloning and Characterization of aBacillus vietnamensisMetalloprotease

“…3). Thermolysin is known to bind four calcium ions to form a ''double'' calcium-binding site and two single-binding sites, 31) whereas for BVMP, the observed calcium-binding sites are constituted only with E185, Y183, T189, I192, and D195 (Fig. 2).…”

Thermostabilization of Bacillus circulans xylanase: Computational optimization of unstable residues based on thermal fluctuation analysis

“…Once hot spot regions for thermostability were identified by experimental or theoretical approaches, further thermostabilization could be achieved by optimizing the hot spot regions using, for example, combinations (Akasako et al, 1995), saturation mutagenesis (McLachlan et al, 2008), or site-directed mutagenesis (Veltman et al, 1997) of the hot spot residues or their neighboring residues. However, a rapid and effective strategy is necessary to reduce the time and effort in engineering enzymes and here we adopted a computational approach rather than random mutagenesis to optimize the hot spot region of Bcx.…”

Engineering an enzyme to resist boiling

“…Indeed, several stabilizing mutations involving residues in the 55-69 region have been identified. These include Xaa 3 Pro mutations (13,14), the introduction of a salt bridge (15), and the introduction of a disulfide bridge (16). These latter mutations were not based on comparison of the sequences and structures of naturally occurring TLPs, but on rational design.…”