BT_1911 kinetics were determined with slight modifications to previously established methods[47 (link)]. 0.2 nM HSDH was added to pre-warmed 37°C 100 μL reactions containing 5 mM NAD+ in 10 mM MOPS. Bile acid substrates were included in concentrations ranging from 40 μM to 4.5 mM. Change in absorbance at 340 nm was monitored every 80 seconds in flat bottom UV-Star 96 well microplates (Greiner) from a Tecan Infinite F200 Pro plate reader. A standard curve of NADH was included and reaction absorbances were converted to substrate concentration since NADH is generated stoichiometrically with reaction products. Initial velocity data was plotted and Michaelis-Menten parameters were calculated in GraphPad Prism by fitting the data to a nonlinear regression model.