Tanaka_2021_Int.J.Biol.Macromol_170_343

Homologous proteins differ in their amino acid sequences at several positions. Generally, conserved sites are recognized as not suitable for amino acid substitution, and thus in evolutionary protein engineering, non-conserved sites are often selected as mutation sites. However, there have also been reports of possible mutations in conserved sites. In this study, we explored mutable conserved sites and immutable non-conserved sites by testing random mutations of two thermostable proteins, an esterase from Sulfolobus tokodaii (Sto-Est) and a subtilisin from Thermococcus kodakarensis (Tko-Sub). The subtilisin domain of Tko-Sub needs Ca(2+) ions and the propeptide domain for stability, folding and maturation. The results from the two proteins showed that about one-third of the mutable sites were detected in conserved sites and some non-conserved sites lost enzymatic activity at high temperatures due to mutation. Of the conserved sites in Sto-Est, the sites on the loop, on the surface, and far from the active site are more resistant to mutation. In Tko-Sub, the sites flanking Ca(2+)-binding sites and propeptide were undesirable for mutation. The results presented here serve as an index for selecting mutation sites and contribute to the expansion of available sequence range by introducing mutations at conserved sites.