) at a flow rate of 1 ml/min over 30 min was utilised to separate the glucose conjugates from their aglycones. Both options contained 0.01 H3PO4. Each peak on the chromatogram was monitored between 190 and 430 nm. The HPLC situations were described inside the following: IAA, ldetection = 210 nm, ten ?eight solvent A; ICA, IPA, IBA, and NAA, ldetection = 280 nm, ten ?0 solvent A; two,4-dichlorophenoxyacetic acid and picloram, ldetection = 287 nm, 10 ?00 solvent A. The products of auxin conjugates synthesized by recombinant UGT74D1 have been further confirmed by the LC-MS method (Thermo Scientific) like the Surveyor autosampler and MS pump (Thermo-Finnigan, San Jose, CA, USA). The methods and mobile phases had been comparable to HPLC situation except that 0.01 acetic acid as an alternative to 0.01 H3PO4. The mass spectrometer operated in a optimistic electrospray ionization mode with 30 eV and a probe voltage of three.0 kV. The temperature was set to 350uC. The information acquisition and evaluation have been performed with Xcalibur application (version 2.0.6). Table 1. Distinct activity of UGT74D1 toward auxins and related substrates.SubstratesSpecific activity (nkat/mg protein) 0.1760.03 1.2560.01 1.8560.01 2.1760.05 1.1560.01 0.3260.08 NDFigure 3. HPLC and LC-MS analysis of reaction item from IBA. (A) HPLC analysis. 1: the reaction was added with GST protein as manage. 2: the reaction was added with UGT74D1 fusion protein in addition to a new peak (peak b) was made. Peak “a” represents the substrate IBA. (B) LC-MS analysis of peak b. (C) LC-MS analysis of IBA glucose conjugates developed by the catalysis of UGT74E2 which was applied as optimistic handle in this investigation.2′,3′-Dideoxy-5-iodouridine Purity (D) Proposed enzymes and biosynthetic pathway for the synthesis of IBA-glucose ester in the aglycone IBA.3,5-Dichloropyrido[3,4-b]pyrazine web doi:ten.PMID:25023702 1371/journal.pone.0061705.gICA IAA IPA IBA NAA two,4-D PicloramProtein Putification and Enzyme Activity AssayEscherichia coli strain XL1-Blue carrying the expression plasmid of GST-UGT74D1 fusion construct was applied to produce the fusion protein. Soluble recombinant protein was induced and purified based on the methods described by Hou et al. [42].Note: The assay mix (100 ml) contained 2 ug of purified UGT74D1 fusion protein, five mM UDP-glucose, 1 mM hormone, 50 mM HEPES (pH 7.0), two.5 mM MgSO4, ten mM KCl and 14.4 mM 2-mercaptoethanol. The reactions had been carried out at 37uC for three h. The results represent the means 6S.D from 3 independent measurements. The precise enzyme activity was defined as nmol of substrates converted into glucose conjugates per second (nanokatal, nkat) by 1 mg of protein. doi:10.1371/journal.pone.0061705.tPLOS One | plosone.orgUGT74D1 Novel Auxin GlycosyltransferaseFigure 4. HPLC evaluation of reaction products from other auxins. 1: the reaction was added with GST protein as control. 2: the reaction was added with UGT74D1 fusion protein. Peak “a” represents the auxin substrates. Peak “b” represents the reaction merchandise. doi:10.1371/journal.pone.0061705.gFactors Affecting the Activity of Recombinant UGT74DBecause UGT74D1 has the highest enzyme activity toward IBA, we pick out the IBA in this study as substrate for analyzing the things affecting the enzyme activity. The calculation of enzyme activity was according to the reduction of peak location in the substrate IBA just before and following reaction. Elements tested consist of temperature, buffer and pH. Each of the reaction mix (100 ml) contained 0.two ug of recombinant UGT74D1, 5 mM UDP-glucose, 1 mM IBA, 2.five mM MgSO4, 10 mM KCl, 14.4 mM 2-mercaptoethanol.
