N-Acetyl-L-glutamate synthase catalyzes the conversion of AcCoA and glutamate to CoA and N-acetyl-L-glutamate (NAG), the first step of the arginine biosynthetic pathway in lower organisms. In mammals, NAG is an obligate cofactor of carbamoyl phosphate synthetase I in the urea cycle. We have previously reported the structures of NAGS from Neisseria gonorrhoeae (ngNAGS) with various substrates bound. Here we reported the preparation of the bisubstrate analog, CoA-S-acetyl-L-glutamate, the crystal structure of ngNAGS with CoA-NAG bound, and kinetic studies of several active site mutants. The results are consistent with a one-step nucleophilic addition-elimination mechanism with Glu353 as the catalytic base and Ser392 as the catalytic acid. The structure of the ngNAGS-bisubstrate complex together with the previous ngNAGS structures delineates the catalytic reaction path for ngNAGS.
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