Fig. 3 | Nature Communications

Fig. 3

From: Increased glutarate production by blocking the glutaryl-CoA dehydrogenation pathway and a catabolic pathway involving l-2-hydroxyglutarate

Fig. 3

Purification and characterization of LhgO. a SDS–PAGE of expression and purification steps of LhgO. Lane M, molecular weight markers; lane 1, crude extract of E. coli BL21(DE3) harboring pETDuet-lhgO; lane 2, the unbound protein of the HisTrap HP column; lane 3, purified LhgO using a HisTrap column. b Substrate specificity of LhgO in P. putida KT2440. Data shown are mean ± s.d. (n = 3 independent experiments). c The time course of reactions of LhgO with l-2-HG, d-2-HG and CsiD-catalyzed product. Sample, CsiD-catalyzed product. d Specific activities of LhgO with different electron acceptors. Data shown are mean ± s.d. (n = 3 independent experiments). e HPLC chromatograms illustrating the product-forming behavior of LhgO in P. putida. Black line, the reaction mixture with denatured LhgO; red line, the reaction mixture with active LhgO. nRIU, nano-refractive index units. f The activities of LhgO and YgaF with molecular oxygen and the effects of FAD and FMN. LhgO and YgaF (1 mg mL−1) were incubated with FAD or FMN (LhgO + FAD, LhgO + FMN, YgaF + FAD, YgaF + FMN) for 1 h just before measurement. The assay mixture contained 50 mM Tris-HCl (pH 7.4) and 5 mM l-2-HG at 30 °C at 900 rpm. After monitoring the background for about 2 min, the enzyme was added, and the oxygen traces were monitored

Back to article page