, 2006). Reaction mixture I (50 μL) contained 100 mM HEPES (pH 7.0), 10 mM α-ketoglutarate, 0.5 mM FeSO4·7H2O, 0.5 mM ascorbate, variable concentrations (0.3–40 mM) of l-leucine, l-threonine or l-methionine, and aliquots of purified dioxygenase. Reaction I was incubated at 30 °C for 30 min, at which point it was arrested by placement on ice. The amount of enzyme applied

was selected to ensure that the increase in synthesized succinate was linear AZD6244 order during the reaction. To determine the concentration of the synthesized succinate, 2.5 μL of reaction mixture I was added to reaction mixture II (up to final volume of 25 μL), which contained 100 mM Tris–HCl (pH 8.0), 1 mM phosphoenolpyruvate, 0.3 mM NADH, 10 mM MgCl2, 0.3 mM CoA, 0.3 mM ATP, 3 μg succinyl-coenzyme A synthetase Venetoclax purchase E. coli (purified by IMAC as his6-tag-fused protein) and 0.25 μL of a solution of pyruvate kinase (PK)/lactate dehydrogenase (LDH) from rabbit muscle (Sigma) (0.186 U of PK and 0.226 U of LDH). Reaction II was incubated at 30 °C for 1 h and halted by placement on ice. Subsequently, the absorbance at 340 nm was measured, and the concentration of synthesized succinate was deduced from a calibration curve obtained by performing reaction

II with succinate standards. Enzymatic activity was quantified by measuring the amount of succinate produced per minute and per milligram of enzyme. The KM and Vmax parameters with standard errors for l-leucine, l-threonine and l-methionine were deduced from Michaelis–Menten kinetic equation plots obtained from nonlinear regression analysis of experimental data using SigmaPlot (http://www.systat.com). The preparation and identification of l-methionine sulfoxide and hydroxylated l-leucine was performed as previously described (Hibi et al., 2011). A biomass sample of BL21(DE3) [pET-HT-BPE] strain from fresh-made LB-agar plates was inoculated into 400 mL of LB broth (2 × 200 mL) supplemented with Ap

(100 mg L−1) and cultivated at 37 °C until A555 nm = 1 was reached. Subsequently, IPTG was added to a final concentration of 1 mM, and the culture was incubated for an additional 2 h. The biomass was harvested by centrifugation and re-suspended in 5 mL of 50 mM HEPES (pH 7) and lysed by one pass through a French press (1000 psi). The five reaction mixtures (2 mL volume) Thiamine-diphosphate kinase then consisted of 25 mM l-threonine, 25 mM α-ketoglutarate, 100 mM HEPES (pH 7), 10 mM FeSO4·7H2O and 1 mL of cell lysate. The reactions were incubated at 37 °C for 15 h with vigorous shaking. Amino acid hydroxylation was monitored by TLC analysis using ninhydrin (2-propanol/acetone/ammonia/water = 25 : 25 : 6 : 4). A 10 mL volume of the resulting supernatant was passed through a 0.22 μm filter and applied to a preparative TLC plate. The hydroxylated l-Thr was collected, eluted with water, freeze-dried and analysed by ESI-MS as described in (Hibi et al., 2011).