Glucose 6 phosphate dehydrogenase

α-D-Glucose 1,6-bisphosphate potassium salt hydrate, α-D-Glucose 1-phosphate disodium salt hydrate, α-D(+)Mannose 1-phosphate sodium salt hydrate, Inosine 5′-monophosphate disodium salt hydrate, Adenosine 5′-triphosphate disodium salt hydrate, Maltose phosphorylase from Enterococcus sp., Fructose-6-phosphate Kinase from Bacillus stearothermophilus, Trimethylamine, 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma-Aldrich (Sigma-Aldrich, Milan, Italy). Trypsin was purchased from ICN Pharmaceuticals (MP Biomedicals Germany GmbH).
AG1x8 Resin 200-400 Mesh Hydroxide Form was purchased from Bio-Rad (Bio-Rad Laboratories Srl, Milan, Italy).
Sypro Orange was from Invitrogen Molecular Probes (Invitrogen Molecular Probes, Monza, Italy), StepOne^TM Real Time PCR System from Applied Biosystems (Applied Biosystems, Foster City, CA, USA), strips from Sarstedt (Multiply–μStrip Pro 8-strip low profile) (Sarstedt Srl, Milan, Italy).
D(+)Maltose monohydrate, β-Nicotinamide adenine dinucleotide phosphate sodium salt, Glucose-6-phosphate Dehydrogenase, Creatine phosphate disodium salt thetrahydrate were from Alfa Aesar (Alfa Aesar, Thermo Scientific, Kandel, Germany).

HLMs, (HMMC-PL, Lot. PL050B-B, pooled from 50 donors), and CYP2C9 BACULOSOMES Plus Reagent, rHuman 0.5 nmol (P2378, Lot. 1495735) from Life Technologies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies rabbit anti-cyt b5 (sc-33174) and goat anti-cyt b5 (sc-9513) were supplied by Santa Cruz Biotechnology (Sta. Cruz, CA, USA). Secondary anti-rabbit (Invitrogen 31460) and anti-goat (Sigma A5420) antibodies conjugated with peroxidase were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Lysozyme, bovine pancreatic DNAse I, glucose-6-phosphate dehydrogenase, and Terrific Broth were obtained from Thermo Fisher Scientific (Waltham, MA, USA). DEAE-cellulose and Phenyl Sepharose CL-4B were supplied by Sigma-Aldrich (St. Louis, MO, USA). Amplex Red from Molecular Probes (Eugene, OR, USA) was supplied by Thermo Fisher Scientific (Waltham, MA, USA). HPLC columns Chromolith RP-18e 4.6 × 5 mm and reverse phase column Chromolith 18_e 4.6 × 100 mm were obtained from Merck (Darmstadt, Germany). Other chemicals used in this work were analytical grade reagents from Sigma-Aldrich (St. Louis, MO, USA) or Merck Millipore (Darmstadt, Germany).

To analyze the substrate preference of AcbQ, the initial velocity was obtained. A reaction mixture containing final concentrations of 50 mM potassium phosphate buffer (pH 7.0), 25 mM MgCl₂, 2 mM ATP, 2 mM NADP, 2 U hexokinase (Sigma Aldrich), 2 U glucose-6-phosphate dehydrogenase (Alfa Aesar by Thermo Fisher Scientific) and 5 mM substrate each (listed below) was preincubated at 30 °C [29 (link)]. The absorbance at 340 nm was measured by a Tecan Infinite M200 microplate reader (Tecan Group AG, Männedorf, Swiss) with the i-control 10.1 software (Tecan Group AG). When no changes of absorbance A₃₄₀ were observed, 2 µM AcbQ was added to achieve a final assay volume of 200 µL and to initiate the enzyme reaction. The assay was performed at 30 °C for 30 min. G2, G3, G4, G5, G6, Tre, Suc, Acb and Acb-7P were tested as substrates. In addition, combinations of Acb with G2, G3 G4, Suc and Tre, and Acb-7P with G2, G3 and G4, were analyzed. All substrate combinations were measured in triplicate. A calibration curve of glucose standards was prepared to calculate concentrations.