Fig. 3: Biochemical characterizations of AcbR and AcbI.
From: Complete biosynthetic pathway to the antidiabetic drug acarbose
a–d The activity of AcbR with V1P, 1-epi-V1P, V1,7PP, or 1-epi-V1,7PP as substrates incubated with various NTPs. The blank was a reaction mixture lacking AcbR. A: ATP, U: UTP, G: GTP, C: CTP, dT: dTTP; e Partial LC-QTOF/MS EIC chromatograms (negative ion mode) of AcbR reaction with V1P and GTP (in red) and the negative control lacking AcbR (in black). Both chromatograms are the extraction of corresponding calculated exact mass for GDP-valienol (m/z 600.0750 [M-H]–). f Coupling between dTDP4a6dGlc and maltose catalyzed by AcbI; g MS/MS analysis of the product of the AcbI reaction; h ESI( + ) EIC for 4-aminoDGG (m/z 510.1793, [M + Na]+) from AcbI reactions with maltose, maltotriose, maltotetraose, and maltopentaose; and (i) ESI( + ) EIC for 4-aminoDGG (m/z 510.1793 [M + Na]+) from AcbQ, AcbS, and AcbI reactions with maltose. For a–d, error bars indicate standard deviation (SD) (n = 3 analytical replicates), and data are presented as mean values ± SD. All experiments were carried out at least three times with similar results. ESI, electrospray ionization mass spectrometry; EIC, extracted ion chromatogram; V1P, valienol 1-phosphate; V1,7PP, valienol 1,7-diphosphate; 1-epi-V1,7PP, 1-epi-valienol 1,7-diphosphate; A, ATP; U, UTP; G, GTP; C, CTP; dT, dTTP; dTDP-4a6dGlc, dTDP-4-amino-4,6-dideoxyglucose; 4-aminoDGG, O-4-amino-(4,6-dideoxy-α-D-glucopyranosyl)-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose.
