Fig. 2: Biochemical characterization of the kinase AcbU and the phosphatase AcbJ.
From: Complete biosynthetic pathway to the antidiabetic drug acarbose
a ESI (–) EIC (m/z 334.9940) of AcbU reaction with V7P; b ESI (–) EIC (m/z 334.9940 and 255.0275) of AcbU reaction with V1P; c ESI (–) EIC (m/z 255.0275) of AcbU reaction with V; d PK/LDH coupled enzyme assay of AcbU reactions with V7P, V, or V1P as substrates, and no substrate (NS) in the presence of ATP. Reaction with boiled AcbU was used as a blank; e PK/LDH coupled enzyme assay of AcbU reactions with V7P, V, or V1P as substrates in the presence of ATP. Reactions of AcbU without substrate but with ATP were used as blanks; f MESG assay of AcbJ reaction with V1,7PP, 1-epi-V1,7PP, V7P, or Aca7P as substrates; g ESI (–) EIC (m/z 255.0275) of AcbJ reactions with V1,7PP. For d–f, 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; V, valienol; V1P, valienol 1-phosphate; V7P, valienol 7-phosphate; V1,7PP, valienol 1,7-diphosphate; 1-epi-V1,7PP, 1-epi-valienol 1,7-diphosphate; Aca7P, acarbose 7-phosphate.
