Three-dimensional structure of Escherichia coli asparagine synthetase B: Ashort journey from substrate to product

Asparagine synthetase B catalyzes the assembly of asparagine from aspartate , Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structu re of the enzyme from Escherichia coli determined and refined to 2.0 Angstr om resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP, Each subunit of the dimeric protein folds into two disti nct domains. The N-terminal region contains two layers of antiparallel beta -sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Ke y side chains employed for positioning the glutamine substrate within the b inding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal do main, responsible for the binding of both Mg(2+)ATP and aspartate, is domin ated by a five-stranded parallel beta-sheet flanked on either side by alpha -helices. The AMP moiety is anchored to the protein via hydrogen bonds with O-gamma of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two acti ve sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensi onal architecture of the N-terminal domain of asparagine synthetase B is si milar to that observed for glutamine phosphoribosylpyrophosphate amidotrans ferase while the molecular motif of the C-domain is reminiscent to that obs erved for GMP synthetase.