Subtilisin evolution

All the four essential features of the active site of chymotrypsin are thus also present in subtilisin. Furthermore, these features are spatially arranged in the same way in the two enzymes, even though different framework structures bring different loop regions into position in the active site. This is a classical example of convergent evolution at the molecular level. 

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Wells, J.A., et al. On the evolution of specificity and catalysis in subtilisin. Cold Spring Harbor Symp. Quant. Biol. 52 647-652, 1987. 

Mammalian PCs, just like kexin, cleave their substrates carboxy-terminal of paired basic residues and they share a conserved catalytic domain resembling that of bacterial subtilisins. The catalytically important residues Asp, His, and Ser are arranged in the catalytic triad in a way that is typical for subtilisins but distinct from the arrangement found within the (chymo)trypsin clan of serine proteases. The subtilisins and (chymo)trypsins have thus served as a prime example of convergent evolution [140,141],... 

L. You and F. H. Arnold, Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylform-amide, Protein Eng. 1996, 9, 77-83. 

Although the comparisons of protein folds can yield valuable insights into protein function and evolution, it is also very desirable to be able to detect structural similarities at the residue or atomic level. This is because the detection of similar patterns of functional groups in different proteins may allow analogies to be drawn between disparate proteins modes of action. The classic example of this are the similar clusters of three catalytic residues in the otherwise unrelated subtilisin and chymotrypsin families of enzymes [82]. 

At present, 16 cysteine-containing subtilisin-type enzymes are known and the position of the cysteine residues is restricted to the nine corresponding sites described above.42 Of the 16 enzymes, six enzymes other than aqualysin I and proteinase K have cysteine residues at positions where the cysteine residues are able to form disulfide bond(s) like the two enzymes. Although these disulfide bonds seem to have been acquired to increase protein stability, only four kinds of disulfide bonds are found in the subtilisin-type enzymes, suggesting that the positions of the disulfide bonds have been selected strictly in the process of molecular evolution of the enzyme. 

Fig. 13. The predicted entropy distribution for subtilisin E as determined by a mean-field treatment of the structural model. When all amino acids are equally allowed at a position, ij = In 20 3.0. The red lines are the positions at which mutations discovered by directed evolution improved the thermostability. The blue lines are for mutations that improved the activity (hydrolysis of a peptide substrate) in aqueous dimethylformamide. The bars indicate the average and standard deviation of the structural entropies.

We chose subtilisin E to test our prediction that directed evolution makes mutations at uncoupled positions (Voigt et al., 2000b). Directed evolution increased the temperature optimum for activity, T pt, of Bacillus subtilis subtilisin E from 59° to 76°C, with eight mutations (Zhao and Arnold, 1999). In an independent study, thirteen mutations improved the activity toward the hydrolysis of su c c i iivI-A 1 a-A1 a-Pro-Phe- >-nitroanilide (s-AAPF-j Na) in the organic solvent dimethylformamide (DMF). The mutants were found by screening 2000 to 5000 clones from... 

Fig. 10. Sequence alignment of subtilisins S41, SSII, S39, BPN, E, Carlsberg, and thermitase. Thermitase is an homologous subtilisin-like protease from the thermophilic bacterium Thermoactinomyces vulgaris. Residues conserved in four or more of the sequences are shaded. The positions of mutations discovered during the directed evolution of the various subtilisins are indicated above the alignment. E-subtilisin E, F-subtilisin S41, S-subtilisin SSII, B-subtilisin BPN. Active site residues are indicated (A).

In another set of experiments, directed evolution was used to stabilize a psychrophilic subtilisin, S41, isolated from the Antarctic bacterium TA41 (Miyazaki et al., 2000 Miyazaki and Arnold, 1999). S41 conserves the overall subtilisin fold and shares relatively high identity with other subtilisins (Fig. 10), but contains several features not found in the common mesophilic subtilisins, including a highly charged surface, several inserted surface loops, and decreased numbers of salt bridges and aromatic-aromatic interactions (Davail et al., 1994). In common with other enzymes from psychrophilic sources, S41 is less stable at high... 

Fig. 15. Evolution of (a) stability (half-life at 60°C) and (b) activity for subtilisin S41.

Taguchi et al. (1998) used directed evolution to increase the low-temperature activity of the mesophilic subtilisin BPN. Random muta-... 

The first step in setting up a successful directed evolution protocol is the development of an efficient expression system using an appropriate bacterial host. This is not a trivial task, in particular when overexpression is to be coupled to enzyme secretion. Fortunately, some proteins can easily be overexpressed and secreted by using commercially available systems [27 - 29], a prominent example being subtilisin of Bacillus subtilis [30]. However, many enzymes of interest are not amenable to such systems examples include a variety of different lipases from Pseudomonas species. 

The charge relay system is found at the active site of a group of enzymes called serine proteases. They include chymotrypsin, trypsin, a-lytic protease, elastase, and subtilisin. It is interesting that the charge relay system was found in enzymes belonging to different branches of diemical evolution (chymotrypsin and subtilisin). This suggests that this system is a hydrolytic catalytic system of general importance which is derived solely from amino acid residues. 

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