Extended Data Fig. 2: In vitro characterization of PolE as an unusual Fe²⁺/pterin-dependent desaturase.

a, Schematic of the reaction catalyzed by PolE. b, PolE reactions with individual addition of BH4, 6-MePH4, and THF. Std, the authentic l-isoleucine and 2 standards; +BH4, the reaction with Fe²⁺, BH4, and FolA added; +6-MePH4, the reaction with Fe²⁺, 6-MePH4, and FolA added; +THF, the reaction with Fe²⁺, THF, and FolA added; NC, the reaction without PolE added as negative control. c, Evaluation of the metal dependence of PolE. PolE was firstly chelated with EDTA, and then its reactions were individually supplemented with various divalent metal ions. d, Schematic of the hypothetic catalytic-process by PolE. e, LC-HRMS analysis of the PolE reactions using 3 or 4 as substrate. Std, the authentic 2, 3 and 4 standards; +(3+PolE), the PolE reaction using 3 as substrate; +3, the reaction without PolE added as negative control; +(4+PolE), the PolE reaction using 4 as substrate; +4, the reaction without PolE added as negative control. f, LC-HRMS analysis of the PolE reactions using 5 or 6 as substrate. Std, the authentic 2, 5 and 6 standards; +(5+PolE), the PolE reaction using 5 as substrate; +5, the reaction without PolE added as negative control; +(6+PolE), the PolE reaction using 6 as substrate; +6, the reaction without PolE added as negative control.