This study examined the roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA).
Partial Order Optimum Likelihood (POOL) was used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5–5, RA95.5–8, and RA95.5–8F, representing progress along an evolutionary trajectory with increasing activity, was examined. Computed measures of coupling between charged states of residues showed that as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, were tested using site-directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants showed decreases in kcat relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibited significant decreases in kcat/KM across the series. Only modest decreases in kcat/KM were observed for the E85Q variants with little effect on kcat. Computed metrics of the degree of coupling between protonation states rose significantly as evolution proceeded and catalytic turnover rate increased. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst. (publisher abstract modified)