Active Site Analysis of the Potential Antimicrobial Target Aspartate Semialdehyde Dehydrogenase

Abstract

Aspartate-beta-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the biosynthetic pathway through which bacteria, fungi, and the higher plants synthesize amino acids, including lysine and methionine and the cell wall component diaminopimelate from aspartate. Blocks in this biosynthetic pathway, which is absent in mammals, are lethal, and inhibitors of ASADH may therefore serve as useful antibacterial, fungicidal, or herbicidal agents. We have determined the structure of ASADH from Escherichia coli by crystallography in the presence of its coenzyme and a substrate analogue that acts as a covalent inhibitor. This structure is comparable to that of the covalent intermediate that forms during the reaction catalyzed by ASADH. The key catalytic residues are confirmed as cysteine 135, which is covalently linked to the intermediate during the reaction, and histidine 274, which acts as an acid/base catalyst. The substrate and coenzyme binding residues are also identified, and these active site residues are conserved throughout all of the ASADH sequences. Comparison of the previously determined apo-enzyme structure [Hadfield et al. J. Mol. Biol. (1999) 289, 991-1002] and the complex presented here reveals a conformational change that occurs on binding of NADP that creates a binding site for the amino acid substrate. These results provide a structural explanation for the preferred order of substrate binding that is observed kinetically.

Aspartate Dehydrogenase, a Novel Enzyme Identified from Structural and Functional Studies of TM1643

“…However, it is unlikely to be an ASA-DH because its active site lacks the Cys residue required for the catalysis by ASA-DH (19). Our enzymatic studies with purified TM1643 showed no ASA-DH activity in either the forward or the reverse reaction.…”

“…This residue is strictly conserved among all family members and may be located near the C4 position of the nicotinamide ring in the closed form of the enzyme. In support of this observation, the His-193 residue is structurally equivalent to the catalytic His residue in ASA-DH (19).…”

“…The iminoaspartate product is unstable in aqueous solution and can decompose to oxaloacetate and pyruvate, as indicated by the reactions in the square brackets. (18), and ASA-DH (19). The amino acid sequence identity among the structurally equivalent residues is below 15%, underscoring the lack of sequence homology between TM1643 and other proteins.…”

Capture of an intermediate in the catalytic cycle of l -aspartate-β-semialdehyde dehydrogenase

“…The structures of ASADHs from Escherichia coli (8,9) and from Vibrio cholerae (10) were recently solved, both as the apoenzyme and as a ternary complex with NADP and a covalently bound active site inhibitor, S-methyl-L-cysteine sulfoxide (SMCS). Each of these structures has further clarified the role of several active site amino acids identified earlier by site-directed mutagenesis (11).…”

Crystal Structure of the LysY·LysW Complex from Thermus thermophilus

“…5A). Interdomain movement upon the binding of NADP ϩ was reported in aspartate-␤-semialdehyde dehydrogenases (28,29); however, this was not the case for TtLysY. A comparison of the present TtLysY structure with the apo-TtLysYHB8 structure revealed that both structures were essentially the same (root mean square deviation ϭ 0.4 Å).…”