A Pair of Promiscuous Surrogates for N-Acetyl-l-Glutamate Synthase, the Initial Enzyme of Bacterial Arginine Biosynthesis.

Abstract

The identification and characterization of promiscuous enzymes is significant because they provide the raw materials for evolving new catalysts and assembling new metabolic pathways. Here, we report the discovery of two promiscuous enzymes from Escherichia coli capable of synthesizing N-acetyl-l-glutamate, the first metabolic intermediate in bacterial arginine biosynthesis. The catalytic subunit of aspartate transcarbamoylase (PyrB) and p-aminobenzoyl-glutamate hydrolase (AbgAB) catalyze the acetyl phosphate-dependent acetylation of l-glutamate with catalytic efficiencies (k_cat/K_{m, glutamate}) of 0.089 and 0.59 M^-1 s^-1, respectively. Although the promiscuous activities of PyrB and AbgAB are over 10⁵ -fold lower than the native E. coli N-acetyl-l-glutamate synthase (ArgA), both can function via multicopy suppression to restore growth to an argA-deficient auxotroph on glucose minimal medium. We also describe a pair of spontaneous chromosomal mutations in the acetate kinase gene that facilitate PyrB-mediated replacement of ArgA. These nonsynonymous substitutions encode two variants, D248E and L279Q, that display ∼130-fold and ∼920-fold reductions in acetate kinase activity, respectively. Past work demonstrates that inactivation of acetate kinase leads to intracellular accumulation of acetyl phosphate, suggesting the observed mutations function by boosting the concentration of this noncanonical PyrB substrate. This work provides the first report of promiscuous N-acetyl-l-glutamate synthases and demonstrates how secondary, loss-of-function mutations can tune metabolism such that previously irrelevant latent activities may be elevated from physiological obscurity to an essential role in biosynthesis following overproduction.