Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase

Abstract

Glucosamine-6P synthase catalyzes the synthesis of glucosamine-6P from fructose-6P and glutamine and uses a channel to transfer ammonia from its glutaminase to its synthase active site. X-ray structures of glucosamine-6P synthase have been determined at 2.05 Å resolution in the presence of fructose-6P and at 2.35 Å resolution in the presence of fructose-6P and 6-diazo-5-oxo-L-norleucine, a glutamine affinity analog that covalently modifies the N-terminal catalytic cysteine, therefore mimicking the γ- glutamylthioester intermediate formed during hydrolysis of glutamine. The fixation of the glutamine analog activates the enzyme through several major structural changes: 1) the closure of a loop to shield the glutaminase site accompanied by significant domain hinging, 2) the activation of catalytic residues involved in glutamine hydrolysis, i.e. the α-amino group of Cys-1 and Asn-98 that is positioned to form the oxyanion hole, and 3) a 75° rotation of the Trp-74 indole group that opens the ammonia channel. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.