Fig. 2: Mutations near FAD are critical for a gain in oxygen reactivity.
From: Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme
a, One hundred and thirty-three variant sequences were isolated from our selection. The observed percentage of missense mutations at each amino acid ___location along the protein’s sequence is plotted. Note that these variant sequences are not independent because the iterative mutagenesis used to create new mutant libraries was done using a pool of higher-activity variants as a template and is therefore subject to a founder effect between different generations of the selection. The color scheme in a applies to the rest of the figure. b, The crystal structure of wild-type NicA2 (Protein Data Bank (PDB) ID 5TTJ) is displayed with a tunnel diameter of ~1.4 Å identified by CAVER simulation rendered in magenta43. c, kcat values determined for single mutations in the background of wild-type NicA2. The dotted line indicates the kcat value of wild-type NicA2. d, kcat values determined for variants where mutations were removed from the background of NicA2 v320; each line represents the kcat value corresponding to single mutations back toward the wild-type NicA2. Note the tenfold difference in scale from the plot shown in c. The dashed line indicates the kcat value of NicA2 v320. The ___location of the amino acid positions L449 and T319 in the crystal structure can be seen in Extended Data Fig. 4.
