Wild-type and mutant PutA from Bradyrhizobium japonicum (BjPutA) in the pKA8H vector were expressed and purified as previously described [59 ]. The His tag was removed from both enzymes as previously described [59 ]. The R51E mutant variant of BjPutA was generated from the pKA8H-BjPutA construct using the QuikChange II XL kit (Agilent). The purified proteins were dialyzed overnight against a storage buffer containing 50 mM Tris (pH 7.8), 50 mM NaCl, 0.5 mM Tris(2-carboxyethyl)phosphine, and 5% (v/v) glycerol.
Assessment of the coupled PRODH-GSALDH activity of both wild-type and R51E BjPutA was carried out as previously described [26 (link)]. Briefly, NADH formation was monitored at 340 nm in assays performed at room temperature in an Epoch 2 plate reader (BioTek). The reaction mixture contained BjPutA or BjPutA R51E (0.5 μM), proline (2.5 – 400 mM), coenzyme Q1 (0.1 mM), and NAD+ (0.2 mM) in a buffer containing 50 mM potassium phosphate, 25 mM NaCl, and 10 mM MgCl2 at pH 7.5 with a final reaction volume of 200 μL. Reactions were performed in the presence and absence of NAD+ to correct for CoQ1 reduction. Linear regression in Origin 2017 was used to determine rates for final analysis. The rate data were fit to a substrate inhibition model in Origin 2017.
Assessment of the coupled PRODH-GSALDH activity of both wild-type and R51E BjPutA was carried out as previously described [26 (link)]. Briefly, NADH formation was monitored at 340 nm in assays performed at room temperature in an Epoch 2 plate reader (BioTek). The reaction mixture contained BjPutA or BjPutA R51E (0.5 μM), proline (2.5 – 400 mM), coenzyme Q1 (0.1 mM), and NAD+ (0.2 mM) in a buffer containing 50 mM potassium phosphate, 25 mM NaCl, and 10 mM MgCl2 at pH 7.5 with a final reaction volume of 200 μL. Reactions were performed in the presence and absence of NAD+ to correct for CoQ1 reduction. Linear regression in Origin 2017 was used to determine rates for final analysis. The rate data were fit to a substrate inhibition model in Origin 2017.
