Ad 14c nad

Heavy D-Glucose (U-¹³C₆, 99% and U-¹³C₆, 99%; 1, 2, 3, 4, 5, 6, 6-²H₇, 97-98%), ¹⁵NH₄Cl (¹⁵N, 99%) and 99.8% D₂O were purchased from Cambridge Isotope Laboratories Inc. [Ad-¹⁴C]-NAD⁺ (279.0 mCi/mmol) and [³H]-formate sodium salt (5 Ci/mmol) were purchased from PerkinElmer and Moravek Biochemicals, respectively. All other chemicals were purchased from Sigma-Aldrich unless otherwise stated. All the reagent concentrations refer to final concentrations in the reaction mixture, unless otherwise specified.

The primers for active site variants were prepared by Integrated DNA Technologies. QuikChange II Site-Directed Mutagenesis Kits were purchased from Agilent Technologies. E. coli BL21 (DE3) pLysS cells were from Novagen. Blue sepharose 6 fast flow and Superdex 200 resin were from GE Healthcare Life Sciences. Bradford dye reagent, SDS gels and the protein standards were from Bio-Rad. [Ad-¹⁴C]-NAD⁺ was from PerkinElmer. [³H]- formic acid was from Moravek Biochemicals. All other materials were purchased from Sigma-Aldrich unless otherwise specified.
Site directed mutagenesis was performed on the gene for WT FDH, using standard procedures, and the primer design is listed in the SI. Plasmids were transformed into BL21 (DE3) pLysS cells and grown in 6 L Luria-Bertani medium with 100 mg/L ampicillin at 37°C and 250 rpm. Expression and purification of WT and variant FDHs were carried out using the procedure in Ref. 29 (link).

The pET-23a plasmid harboring the gene encoding for CbFDH was a generous gift from Dr. Nikolaos Labrou of the Agricultural University of Athens. E. coli BL21 (DE3) pLysS cells were from Novagen. Blue sepharose 6 fast flow and Superdex 200 resin were from GE Healthcare Life Sciences. Bradford dye reagent, immobilized pH gradient (IPG) strips for isoelectric focusing (IEF), SDS gels and the protein standards were from Bio-Rad. [Ad-¹⁴C]-NAD⁺ was from PerkinElmer. [³H]-formic acid was from Moravek Biochemicals. All other materials were purchased from Sigma-Aldrich unless otherwise specified.