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The newly created R-enantioselectivity of the esterase mutein (ERapp = 1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site.
Sulfolobus acidocaldarius β-glycosidase (BGAL_SULAC) was used as an extremophilic enzyme model to study the effect of mutations close to the catalytic residues on the enzyme activity and the pH-activity profile.
Only mutations close to genes are shown (see Materials and methods for details).
Additionally, point mutations close to breakpoint junctions are subjected to further examination during Sanger confirmation.
Interestingly, Case#1 and #2 with homozygous mutations close to and within the ARM region, respectively, presented microcephaly (Table 1).
In fact, the distribution in the alignment used combinations of the mutations close to the binomial distribution.
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The result also supported the hypothesis that underlying these haplotype frameworks were older mutations closer to the root of the gene tree.
Interestingly, the genotype effects on IMCL accumulation increased in significance with mutations closer to the coding regions of the UCN3 gene (Table 1).
Analyses of intracellular COL4A1 and COL4A2 demonstrated a trend of increased intracellular COL4A1 and COL4A2 for mutations closer to the carboxy end of this domain.
Furthermore, molecular dynamics simulations showed that mutations closest to H266, such as Y195A, had the greatest effects on preventing the rotation of helix 3.
Mutations closer to the C-terminal had severe effects on the activity and in addition many of the mutations had decreased solubility (as seen for TH p.Gly414Arg, p.Arg441Pro, p.Gln459*, p.Pro492Leu, p.Asp498Gly, and p.Leu510Gln).
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