Engineering a more specific E. coli glyoxylate/hydroxypyruvate reductase for coupled steady state kinetics assays

Abstract

The E. coli glyoxylate reductase/hydroxypyruvate reductase A (EcGhrA) was investigated as a coupling enzyme to monitor the transamination of 2-ketoarginine and glycine by the L-enduracididine biosynthetic enzyme MppQ. Surprisingly, 2-ketoarginine proved to be an efficient substrate for EcGhrA. Since the promiscuity of EcGhrA prevented its use as a coupling enzyme to monitor the aminotransferase activity of MppQ, we set about engineering a more specific variant. X-ray crystal structures of EcGhrA were determined in the unliganded state, as well as with glyoxylate and 2-ketoarginine bound. The electron density maps of EcGhrA with 2-ketoarginine bound showed weak electron density for the side chain of this substrate, complicating the choice of active site residues to target for site-directed mutagenesis.