Capture of an intermediate in the catalytic cycle of L-aspartate-beta-semialdehyde dehydrogenase

Abstract

The structural analysis of an enzymatic reaction intermediate affords a unique opportunity to study a catalytic mechanism in extraordinary detail. Here we present the structure of a tetrahedral intermediate in the catalytic cycle of aspartate-beta-semialdehyde dehydrogenase (ASADH) from Haemophilus influenzae at 2.0-A resolution. ASADH is not found in humans, yet its catalytic activity is required for the biosynthesis of essential amino acids in plants and microorganisms. Diaminopimelic acid, also formed by this enzymatic pathway, is an integral component of bacterial cell walls, thus making ASADH an attractive target for the development of new antibiotics. This enzyme is able to capture the substrates aspartate-beta-semialdehyde and phosphate as an active complex that does not complete the catalytic cycle in the absence of NADP. A distinctive binding pocket in which the hemithioacetal oxygen of the bound substrate is stabilized by interaction with a backbone amide group dictates the R stereochemistry of the tetrahedral intermediate. This pocket, reminiscent of the oxyanion hole found in serine proteases, is completed through hydrogen bonding to the bound phosphate substrate.

Fig. 1.

Stereoview of a surface representation colored according to electrostatic potential, such that regions with potential <–10 k_BT are red, and those >+10 k_BT are blue (k_B, Boltzmann constant; T, absolute temperature). The active sites are located in broad clefts, with the cleft on the front of the dimer annotated.

Fig. 2.

(A) Stereoview of the (2F_o–F_c) electron density map, contoured at 1.3σ, of the active site of hiASADH with the hemithioacetal derived from ASA covalently bound to active site Cys-136. (B) Stereoview of the (2F_o–F_c) electron density map, contoured at 1.2σ, of the active site of the hiASADH with the covalent hemithioacetal and two molecules of phosphate.

Fig. 3.

A comparison of the interactions and orientations between key active site residues and the intermediate complexes. (A) Structure of the ASADH–SMCS complex. (B) Structure of the ASADH–hemithioacetal complex. (C) Model of the acyl-enzyme intermediate, showing the likely repositioning of the oxygen after oxidation by NADP and the proposed attack by phosphate.

Fig. 4.

Schematic representation of the ASADH active site with a bound hemithioacetal reaction intermediate. Interactions between ASA and phosphate and key active site residues are shown.

Fig. 5.

Superimposition of NADP from the V. cholerae ASADH/NADP complex onto the hiASADH/ASA active site, showing the orientation of NADP relative to the tetrahedral intermediate. The substrate oxygen is pointed away from NADP in a position that would facilitate removal of the hydrogen, with the proposed trajectory for hydride transfer shown (· · ·).

Fig. 6.

Proposed mechanism for the catalytic cycle of ASADH shown in reverse biological direction. (A) Tetrahedral intermediate derived from reaction with ASA. (B) Proposed acyl intermediate with trigonal planar geometry. (C) Second tetrahedral intermediate with covalently bound aspartyl phosphate.