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

Abstract

ABSTRACTThe 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. The structure of the complex did, however, suggest that the side chain of W45 could interact with the guanidinium group of 2-ketoarginine. We therefore generated the EcGhrAW45F variant and tested it for activity with 2-ketoarginine, glyoxylate, oxaloacetate, α-ketoglutarate, α-oxofuranacetic acid, phenyl pyruvate, 3-mercaptopyruvate and 2-ketobutyric acid. The W45F variant exhibited a ∼10-fold decrease in the specificity constant (kcat/KM) for 2-ketoarginine, while the reaction with glyoxylate was not significantly impaired. The reactions of the W45F variant with the alternative substrates oxaloacetate and α-ketoglutarate were also impaired. Thus, the W45F variant is a less promiscuous enzyme than the wild-type. This engineered EcGhrAW45F variant could be generally useful as a coupling system for enzymes that produce glyoxylate, such as 4-hydroxy-2-oxoglutarate aldolase or isocitrate lyase.