Purification, crystallization and preliminary X-ray analysis of aspartokinase III fromEscherichia coli

Abstract

Aspartokinase III catalyzes the commitment step in the aspartate metabolism pathway, the phosphorylation of aspartic acid. The Escherichia coli enzyme has been crystallized in the presence of its natural substrate (aspartic acid) and Mg-ADP and diffraction data has been collected at a synchroton source. The crystals belong to the orthorhombic space group C222 1 , with unit-cell parameters a = 60.44, b = 190.31, c = 99.55 A Ê , and data 99.3% complete to 2.7 A Ê . Solving the structure of AK III will provide the ®rst structure of an aspartokinase from any organism.

Site-directed Mutagenesis of Escherichia coli Acetylglutamate Kinase and Aspartokinase III Probes the Catalytic and Substrate-binding Mechanisms of these Amino Acid Kinase Family Enzymes and Allows Three-dimensional Modelling of Aspartokinase

“…11 We have used the results of the present study on NAGK mutations to set a reference frame for judging the effects of mutations that we introduce now into the AKIII of E. coli, 7,8 at five residues that we propose, on the basis of sequence alignment with NAGK, to play roles in catalysis and in the binding of the substrates similar to those of their counterparts in NAGK. For the same reason as in the mutagenesis of NAGK, mild amino acid changes have been introduced in the mutant AKIII forms.…”

“…Therefore, it would appear important to gather more information on AK, including information about its 3-D structure. Unhappily, although crystals of AKIII of E. coli (the form of AK involved in lysine synthesis in this microorganism 8 ) have been generated, 11 the crystallographic structure of an AK has not been determined. We reasoned that if we could ascribe to individual AK residues specific roles on substrate binding and catalysis, this information would be of much use toward understanding the function of AK, and it might guide efforts to construct a structural model of the amino acid kinase domain of this enzyme.…”

Structures of R- and T-stateEscherichia coliAspartokinase III

“…Mutational analyses of the C-terminal AKIII region indicate this to be the site for lysine binding (24,25). Until now, crystal structures of AKIII have not been available; a reported crystallization does not apparently yield a structure (26). The lack of AKIII crystal structures has led to attempts at homology modeling of the catalytic domain (11).…”

Prediction of physical protein–protein interactions

“…True macromolecular protein complexes represent only a small fraction of the currently 27 570 entries (October 2004) in the Protein Data Bank (PDB). The PDB [176] has a bias toward proteins that are easy to express, purify and crystallize, and a negative bias toward membrane proteins-and protein-protein complexes. As of October 2004, 18 930 macromolecular assemblies from 29 277 different PDB files (including obsolete ones) are present in the PQS server [168], and this number includes redundant and biologically non-relevant complexes.…”