Table 1 Amino acid substitutions selected for modification of proteinase K.
From: Engineering proteinase K using machine learning and synthetic genes
Substitution | Effect | Reason for Selection |
|---|---|---|
N95C | Lethal | Literature report: disulphide bond between 95C and 299C reported to stabilize subtilisin BPN' (S3C and Q206C in subtilisin)[34,35]. |
P97S | Lethal | Literature report: P to S reported to increase stability in subtilisin BPN' (P5S in subtilisin)[34,35]. |
S107D | Negative | Homolog sequence alignment analysis: D present at this position in 2/42 Group B homologs. |
S123A | Positive | Thermostable homolog sequence alignment analysis: residue found in 8/11 Group C homologs and 6/42 Group B homologs. |
I132V | Positive | Thermostable homolog sequence alignment analysis: residue found in 10/11 Group C homologs, 1/6 Group A homologs and 13/42 Group B homologs. Also a favorable change according to Dayhoff substitution matrix[36]. |
E138A | Lethal | Literature report: acidic residue to A reported to increase stabibility in subtilisin BPN' (D41A in subtilisin)[34,35]. |
M145F | Negative | Literature report: M to F reported to increase stabibility in subtilisin BPN' (M50F in subtilisin) [34,35]. |
Y151A | Strong positive | Thermostable homolog sequence alignment analysis: residue found in close thermostable homolog gi|131084 and 2/42 Group B homologs. |
V167I | Negative | Substitution matrix-derived change: favorable change according to Dayhoff substitution matrix [36]. Residue found in 1/6 Group A homologs and 27/42 Group B homologs. |
L180I | Positive | Thermostable homolog sequence alignment analysis: residue found in 10/11 Group C homologs, 1/6 Group A homologs and 10/42 Group B homologs. Also a favorable change according to Dayhoff substitution matrix [36]. |
Y194S | Negative | Random mutation obtained during synthesis of wt proteinase K. |
A199S | Negative | Substitution matrix-derived change: favorable change according to Dayhoff substitution matrix [36]. Residue found in 1/6 Group A homologs and 9/42 Group B homologs. |
K208H | Positive | PCA identification of amino acids responsible for clustering of thermophilic sequences gi|4092486; gi|56160990; gi|114081 within Group A and B homologs [37]. |
A236V | Lethal | PCA identification of amino acids responsible for clustering of thermophilic sequences gi|4092486; gi|56160990; gi|114081 within Group A and B homologs [37]. |
R237N | Negative | Thermostable homolog sequence alignment analysis: residue found in 9/11 Group C homologs, 1/6 Group A homologs and 1/42 Group B homologs. |
P265S | Negative | Structural considerations: literature report: P5S reported to increase stability in subtilisin BPN' (P5S in subtilisin) [34,35]. 265S found at this position in proteinase K closest homolog (gi|131084). |
V267I | Positive | Substitution matrix-derived change: favorable change according to Dayhoff substitution matrix [36]. Residue found in 1/6 Group A homologs and 1/41 Group B homologs. |
S273T | Positive | Thermostable homolog sequence alignment analysis: residue found in 11/11 Group C homologs, 1/6 Group A homologs and 29/41 Group B homologs. Also a favorable change according to Dayhoff substitution matrix [36]. |
G293A | Strong positive | Thermostable homolog sequence alignment analysis: residue found in 11/11 Group C homologs, 1/6 Group A homologs and 38/41 Group B homologs. |
L299C | Lethal | Disulphide bond between 95C and 299C reported to stabilize serine proteases [34,35]. |
I310K | Negative | Literature report: K substitution at this position reported to increase stabibility by adding hydrogen bonding in subtilisin BPN' (Y217K in subtilisin) [34,35]. |
K332R | Positive | Thermostable homolog sequence alignment analysis: residue found in 8/11 Group C homologs and 1/6 Group A homologs. Also a favorable change according to Dayhoff substitution matrix [36]. |
S337N | Positive | Thermostable homolog sequence alignment analysis: residue found in 8/11 Group C homologs, 1/6 Group A homologs and 2/41 Group B homologs. Also a favorable change according to Dayhoff substitution matrix [36]. |
P355S | Negative | Structural considerations: literature report: P5S reported to increase stability in subtilisin BPN' (P5S in subtilisin) [34,35]. 355S found at this position in proteinase K closest homolog (gi|131084). |