Fig. 3: Functional analysis of PikC mutants.

a The scheme of PikC_H238pAcF catalyzed aglycone hydroxylation reactions. b The relative activities of the mutants with His238 replaced by different ncAAs towards 6 and 7. Data are presented as mean values ± SEM. Black diamond-dots represent individual data points for n  =  3 replicates. Statistical analysis was performed using the Student’s t test (two-tailed; *P < 0.05, **P < 0.01). c–f, HPLC analysis (230 nm) of the enzymatic reactions catalyzed by the wild type (WT) and indicated mutant PikC enzymes using 6 (c), 7 (d), 4 (e), and 5 (f) as substrates. Each individual in vitro enzymatic reaction containing 1 µM PikC (wild type or mutant), 1 mM NADPH, 10 µM Fdx1499, 5 µM FdR0978, and 0.5 mM substrate in 100 μL storage buffer was incubated at 30 °C for 40 min. The peaks marked by asterisk in (d) denote the spontaneous decomposition product of 7.