Mutagenesis and Chemical Rescue Indicate Residues Involved in β-Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B

Abstract

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of ␤-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wildtype AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation ␤-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to ␤-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

Asparagine Synthetase Chemotherapy

“…In this model, PP i was positioned over the pyrophosphatase loop region of the enzyme so that (a) the hydrogen bonding interactions with the enzyme corresponded to those observed in the GMPS/AMP/PP i crystal structure (48), and (b) noncovalent interactions with the active site Mg 2+ ion were maintained. Further MD refinement and energy minimization then gave a computational model for the AS-B/βAspAMP/PP i complex ( Figure 5), which was checked for consistency with the results of mutagenesis and kinetic studies employing AS-B (19,34,131,(134)(135)(136). Importantly for inhibitor development, we were able to identify completely conserved residues having side chains positioned within 5 Å of either βAspAMP or PP i .…”

“…Access to recombinant human enzyme, albeit in small quantities, in these experiments did permit limited insights into properties such as substrate specificity (31,32). As a result, until the very recent development of an efficient expression system for human ASNS (vide infra) (33), procedures for obtaining large amounts of the glutamine-dependent ASNS (AS-B) encoded by the asnB gene in Escherichia coli (34) were essential to detailed investigations of the structure and mechanism of the enzyme.…”

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

“…Asparagine synthase converts aspartic acid to asparagine in an ATP-dependent two-step reaction, using glutamine or ammonia as the amine donor (3)(4)(5). OxyD contains the conserved N-terminal nucleophilic cysteine (Cys2) which is involved in the hydrolysis of glutamine (64) as well as the conserved adenylation domain that activates the acid moiety of the amine acceptor (6). We hypothesize that OxyD may therefore amidate either malonylcoenzyme A (malonyl-CoA) or malonyl-ACP to yield malonamyl-CoA or malonamyl-ACP (see Fig.…”

Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

“…Replacement of Arg-36 with lysine had a similar effect as replacement with alanine, suggesting that, in addition to the positive charge at this position, the two-point interaction of the guanido group with Asp-34 is important. Chemical rescue of kinetic parameters with guanido derivatives has been reported for arginine replacement variants of several enzymes (2,8,20,21,27,29), including Cam, the prototype of the ␥-class carbonic anhydrase (39). These derivatives are thought to occupy the cavity vacated by the arginine side chain in a productive conformation which mimics the guanido group of arginine.…”