Leuconostoc mesenteroides

For phosphoglucomutase activity, quantification-coupled enzymatic assay following the reduction of NAD⁺ due to G6P oxidation was performed as described in Lindahl and Florencio (2003) with minor modifications. A total amount of 50–200 µg of extract proteins of the soluble fraction of Synechocystis strains was mixed in buffer with a final concentration of 100 mM Tris–HCl pH 8, 4 mM MgCl₂, 2.5 mM NAD⁺, and 0.2 U of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Sigma) in 200 µl. Absorbance at 340 nm was measured and the reaction was triggered by adding 4 mM G1P and 40 µM G16BP, and followed for 30 min at 30 °C. In parallel, as background control for every sample, the same reaction without G1P addition was performed and the changes in absorbance were subtracted from the reaction with G1P.

Hydrogen peroxide concentrations were measured on the stock solution (Sigma) and the OSB, using an enzymatic assay [86 (link)] with horseradish peroxidase C type VI (Sigma), and 3,3′,5,5′-Tetramethylbenzidine (TMB) (DIAGEN CA), and by estimating the ΔOD_653nm and using the Lambert-Beer equation. Meanwhile, the concentration of MB was spectrophotometrically estimated using the Lambert-Beer equation, as described by Buchholz et al. [35 (link)].
Glucose, galactose, and ammonium concentrations were determined as previously described [33 (link)]. Similarly, G1P, and G6P concentrations were calculated by standard enzymatic methods. G1P was determined using 1 U × mL⁻¹ of phosphoglucomutase (PGM) (from rabbit muscle, Sigma), 1 U × mL⁻¹ of glucose-6-phosphate dehydrogenase (G6PDH) (type XXIV from Leuconostoc mesenteroides, Sigma), and 50 µM glucose-1,6-biphosphate, 0.72 mM NADP⁺. The ΔOD_340nm was used to calculate the G1P concentration using the Lambert-Beer equation and the extinction coefficient of NADPH at 340 nm of 6220 M⁻¹ × cm⁻¹. Meanwhile, the G6P concentration was calculated using the same protocol, but excluding PGM and glucose-1,6-biphosphate from the assays.

3′-NADP purified from in vitro reactions or small metabolite extracts was assayed for its capability to induce activity of d-glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Sigma-Aldrich). Reactions were performed similarly to spectroscopic assays using the PK/LDH system in 10-mm-path length quartz cuvettes in buffer O (50 mm HEPES-NaOH, pH 7.5, 200 mm NaCl). Final concentrations of d-glucose-6-phosphate dehydrogenase (G6P-DH) were 14.7 units/ml. 200 μm 3′-NADP from in vitro reactions or SMEs was added to the reaction after 2 min and then incubated for 10 min. To verify G6P-DH activity, 200 μm 2′-NADP (Roche Diagnostics GmbH) was added after the 10-min incubation period, and the reaction was allowed to proceed until all 2′-NADP was reduced to 2′-NADPH.

Dulbecco’s Phosphate Buffered Saline (DPBS 1X, 14190, Gibco) was adjusted to pH 7.4, and to an osmolarity of 295 ± 5 mOsm using an osmometer (Gonotec OSMOMAT 030) by adding glucose. Dextran (from Leuconostoc mesenteroides, ≈2000 kDa, D5376, Sigma-Aldrich) was solubilized at 9% (wt/wt) in PBS by overnight stirring at 50°C. Dextran solution had a viscosity η_o = 39.2 ± 0.7 10⁻³ Pa·s at room temperature, measured with a cone-plane rheometer (MARSIII Cone C35/2°, Thermo Fisher/Haake) and its density matched RBC density, thus preventing cell sedimentation.

Estimates of sucrase activity were obtained using the method outlined by Akkarachiyasit et al. [17 (link)] with slight modifications. Sucrose and sucrase derived from Leuconostoc mesenteroides were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The reaction mixture comprised 40 μL of sucrose (480 mM) with 10 μL of sucrase (0.33 units/mL) in 50 μL of phosphate buffer solution (100 mM, pH 7.0). The mixtures were incubated at 37 °C for 60 min and then at 100 °C for a further 10 min to stop the reaction. The glucose concentrations released from the reaction mixtures were estimated via the glucose oxidase method (glucose assay kit, Sigma Chemical Co., St. Louis, MO, USA) using a VersaMax microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA) at an absorbance wavelength of 540 nm.