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. 2020 Jun 8;10(14):7512–7525. doi: 10.1021/acscatal.0c01487

Figure 7.

Figure 7

Fitness landscapes in variants derived from PseFDH V9. (a) The introduction of the four additional mutations (D221Q, C255A, H379K, and S380V) into variant A198G yields 4! = 24 possible evolutionary pathways. Thick lines indicate the four possible pathways that can be explored by stepwise evolution with the available double and triple mutants. (b) Variants and the four possible pathways from double mutant A198G/D221Q (GQ) via triple variants A198G/D221Q/C255A (GQA), A198G/D221Q/S380V (GQV), and A198G/D221Q/H379K (GQK) and quadruple mutants A198G/D221Q/C255A/S380V (GQAV) and A198G/D221Q/H379K/S380V (GQKV) toward PseFDH V9 or quintuple mutant A198G/D221Q/C255A/H379K/S380V (GQAKV). (c) Catalytic efficiencies (mM–1 s–1) of the deconvoluted variants (shown in the left) toward either NAD+/formate (orange/yellow) or NADP+/formate (green/blue). The coenzyme specificity ratio (CSR) toward NADP+ ((kcat/KM)NADP+/(kcat/KM)NAD+) is shown in the middle (white). The red lines correspond to the pathway exemplified and described in the main text.