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. 2015 Aug 28;6:8041. doi: 10.1038/ncomms9041

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(a) Schematic representation of the conserved cysteine and cofactor-binding sites of YUC6. The relative locations of a conserved FAD-binding domain (a.a. 34–43; gold hatch), Cys-85 (red asterisk) and NADPH-binging domain (a.a. 202–210; red hatch) are depicted along the full-length ORF of YUC6 (417 a.a.; white bar). The FAD- and NADPH-binding sites and the sequence around Cys-85 of all Arabidopsis YUC proteins were aligned, and the residues that were subjected to site-directed mutagenesis are indicated by red boxes. The actual amino-acid substitutions at each location are indicated. (b) NADPH-dependent TR activity of purified recombinant YUC6 protein in the presence (+Trx, left) or absence (−Trx, right) of Trx-h3. TR activity was measured as using DTNB as a substrate and is expressed as increased absorbance at 412 nm. Trx-h3 was added as a reducer in +Trx. Purified recombinant Arabidopsis NTRC TR domain (NTRC-TR) was used as a positive control. (c) DTT-dependent insulin reductase activity of recombinant YUC6 protein. Insulin reductase activity is expressed as the increase in absorbance at 650 nm. DTT only refers to a sample containing insulin and DTT but no YUC6. The other samples contained the same amount of insulin with increasing amounts of YUC6 in the indicated molar ratios, in addition to DTT. (d) Comparison of the TR activity of WT and mutant YUC6 proteins. TR activity of the purified recombinant YUC6 proteins was measured using DTNB as described above. Relative activity refers the rate of Abs_412nm increase for a sample when the rate of Abs_412nm increase for WT YUC6 is taken as 1. Recombinant NTRC-TR was used as a positive control for TR activity. Data represent the means±s.e., n=3. (e) Comparison of the YUC activity of WT and mutant YUC6 proteins. YUC activity using IPA as a substrate was measured using Ehrlich reagent at Abs_585nm and relative activity were calculated as same as TR activity. Recombinant YUC1 was used as a positive control for YUC activity. Data represent the means±s.e., n=3.