## 2 Materials and Methods
### ADPH Isolation and Purification  
ADPH isolation has been previously reported [25,26], and optimizations of that protocol are described below. Luria-Bertani (LB) media was prepared freshly or within 24 h of use; 2 L portions were made in 4 L flasks. Overnight cultures of _E. coli_ WBB06 (HepI and HepII knockout _E. coli_ strain) were started by inoculating 10 mL of LB-Tet with WBB06 cells from a glycerol stock stored at \(-80\)\({}^{\circ}\)C. A minimum of 8 L of LB-Tet media supplemented with 5 mM CaCl\({}_{2}\)[27] was inoculated with the overnight culture (1 mL of overnight growth per 2 L of media) and allowed to grow at 37 \({}^{\circ}\)C to an OD\({}_{600}\) of 1 (approximately 10-12 h), then centrifuged for 10 min at 5000 rpm to pellet the cells. The cell pellets were scraped into a 250 mL beaker to which a stir bar and 80 mL of 50% ethanol (10 mL of 50% ethanol per 1 L of growth) were added and allowed to stir on ice for 2 h. A total of 50% ethanol was prepared using 200 proof ethanol and pH 8 water in an effort to prevent breakdown of sugar nucleotides at pH's lower than 7. Cellular debris was removed by centrifugation in 30 mL Nalgene tubes at 6000 rpm for 20 min at 4 \({}^{\circ}\)C. The supernatant was decanted into 150-200 mL Erlenmeyer flask with a ground joint neck for Schlenk-line vacuum attachment. The ethanol was evaporated from the supernatant by stirring under vacuum on ice and monitoring changes in volume; it should reduce to about half its original volume in approximately 1 h. The extract was further clarified through ultracentrifugation at 40,000 rpm for 1 h at 4 \({}^{\circ}\)C. The clarified extract was filtered successively using Amicon Ultra-15 30 kDa and 3 kDa centrifugal filters to remove proteins and other large nucleotide species. An additional centrifugal filter at 10 kDa MWCO can be used in between the 30 kDa and the 3 kDa to reduce filtration time. The pH of the flow through was analyzed to ensure it remained at pH 8 for the duration of the extraction process, if not, adjustments should be made at this point. pH adjustments can be made simply by using 0.2 M HCl or 0.2 M NaOH (1 M solutions should not be used to prevent overshooting the solution pH which could be detrimental to the small molecule integrity). The extract was purified by placing it over a 64 mL DEAE column using a triethylamine bicarbonate (pH 8) gradient from 1-500 mM using pH 8 ultrapure water. The purification method was established on an Akta Purifier 10 with a flow rate of 3 mL/min and a pressure setting at 3 MPa (according to manufacturer's recommendations). Fractionation was on a peak dependent setting where peaks were seen from UV traces at 254 nm. Further, 10-15 mL fractions were collected in labelled 15 mL falcon tubes and approximately 500 \(\mu\)L of each fraction were flash frozen and lyophilized. All fractions were stored at \(-80\)\({}^{\circ}\)C until characterization was complete to prevent breakdown. ESI-mass spectrometry was used to determine fractions that contained pure ADPH by observation of a peak at (\(m/z^{-1}\) = 619) in a 50:50 acetonitrile:water solution (Figure S1). Fractions confirmed to contain ADPH were lyophilized, combined into a smaller volume of pH 8 water and re-lyophilized to remove traces of triethylamine. Presence of ADPH can also be determined with a variety of other characterization techniques (discussed below).