In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB

Abstract

Our previously presented method for high throughput computational screening of mutant activity (Hediger et al., 2012) is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). Using an appropriate cutoff criterion for the computed barriers, the qualitative activity of 15 out of 22 mutants is correctly predicted. The method identifies four of the six most active mutants with ≥3-fold wild type activity and seven out of the eight least active mutants with ≤0.5-fold wild type activity. The method is further used to screen all sterically possible (386) double-, triple- and quadruple-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified.

Keywords: Computational Chemistry; Enzyme Engineering.

Figure 1. Reaction scheme for the formation of TI.

Nucleophilic attack by O^γ of S105 on carbonyl carbon C²⁰ of substrate. R₁: −CH₂–Cl,R₂: − CH₂–C₅H₆.

Figure 2. Position of point mutations.

(A) Overlay of mutations W104[F, Q, Y]. (B) Overlay of mutations A141[N, Q]. (C) Overlay of mutations of I189[A, G, H, N, Y]. (D) Mutations P38H, G39A, G41S, T42A, T103G, A132N, L278A, A282G, I285A, V286A. Substrate shown in magenta.

Figure 3. Comparison of experimental and computed activities.

1/−1 correspond to increased/decreased overall activity, respectively. Prediction rate is 15/22(68%).

Figure 4. Barrier scatter plot of set S.

22 mutants; the cutoff value c_S is discussed in the text.

Figure 5. Barrier scatter plots of set L.

In both panels, the labels indicate mutants containing the labeled and possibly additional mutations up to the indicated order. “OTHER” indicates a mutant not containing any of the labeled mutations or of higher than 4 order. (A) Mutations of W104. (B) Mutations of I189.