Structural basis for discrimination between oxyanion substrates or inhibitors in aspartate-β-semialdehyde dehydrogenase

Abstract

The reversible dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde (ASA) in the aspartate biosynthetic pathway is catalyzed by aspartate-beta-semialdehyde dehydrogenase (ASADH). The phosphate that is present to activate the aspartate carboxyl group is held in a separate and distinct binding site once removed and prior to its release from the enzyme. This site had been shown to be selective for tetrahedral oxyanions, with several competitive inhibitors and alternative substrates previously identified for the reverse reaction. Structural studies have now shown that the most potent oxyanion inhibitor (periodate) and a good alternative substrate (arsenate) each occupy the same catalytic phosphate-binding site. However, a rotation of a threonine side chain (Thr137) in the periodate complex disrupts an important hydrogen-bonding interaction with an active-site glutamate (Glu243) that participates in substrate orientation. This subtle change appears to be the difference between a substrate and an inhibitor of this enzyme.

Structural Characterization of Inhibitors with Selectivity against Members of a Homologous Enzyme Family

“…The best refined portion of the density is tetragonal in shape and corresponds to the phosphonate moiety of the inhibitor. This group is bound in the site where the phosphate group required for phosphorylation of the substrate (ASA) in the reverse reaction had previously been shown to bind (23). This anion site is composed of a ‘recognition dyad’ where the phosphonate group is oriented similarly to phosphate through electrostatic interactions with the positively charged side chains of Arg99 and Lys223.…”

Arsenate Replacing Phosphate: Alternative Life Chemistries and Ion Promiscuity

“…The position of the bound arsenate, and those of its protein ligands, are identical for both phosphate and arsenate. Critical hydrogen-bonds with catalytic residues, and with a water molecule ligand, are also maintained (18) (Figure 1). In accordance, arsenate comprises a surprisingly good substrate for this enzyme, both in terms of K M (1.6 mM, versus 2.9 mM for phosphate), and k cat (510 and 710 min −1 ) (14).…”

“…Similar mechanistic approaches are still being used with glycosyltransferases that use phospho-sugars as substrates (25, 26). Arsenate-based structures can provide a glimpse of the nature of reaction intermediates, as demonstrated with PNP (20) (Figure 3), and can also shed some light on the ion binding mode in phosphate-utilizing enzymes such as ASADH (18) (Figure 2). …”

Crystal Structure of N-acetyl-γ-glutamyl-phosphate Reductase from Mycobacterium tuberculosis in Complex with NADP+

“…Crystal structures and the catalytic mechanism of ASADH from a number of organisms have been studied using both native proteins as well as their complexes with the cofactor and substrate. [8][9][10][11][12][13][14] These structures have captured the enzymatic reaction intermediate and have revealed the critical catalytic residues. 10 In particular, the active site cysteine plays a key role, since its S γ atom nucleophilically attacks the carbonyl carbon C γ of β-aspartyl-phosphate.…”