Crystal Structures of the Iron-Sulfur Cluster-Dependent Quinolinate Synthase in Complex with Dihydroxyacetone Phosphate, Iminoaspartate Analogues, and Quinolinate

Abstract

The quinolinate synthase of prokaryotes and photosynthetic eukaryotes, NadA, contains a [4Fe-4S] cluster with unknown function. We report crystal structures of Pyrococcus horikoshii NadA in complex with dihydroxyacetone phosphate (DHAP), iminoaspartate analogues, and quinolinate. DHAP adopts a nearly planar conformation and chelates the [4Fe-4S] cluster via its keto and hydroxyl groups. The active site architecture suggests that the cluster acts as a Lewis acid in enediolate formation, like zinc in class II aldolases. The DHAP and putative iminoaspartate structures suggest a model for a condensed intermediate. The ensemble of structures suggests a two-state system, which may be exploited in early steps.

Scheme 1.

NadA catalyzed reaction.

Figure 1.

Stereo diagrams (left) and schematic drawings (right) of the active site of PhNadA with bound (A) DHAP, (B) itaconate, and (C) QA. Black broken lines denote potential hydrogen bonds, red spheres denote waters, and the green sphere denotes chloride.

Figure 2.

(A) Superimposition of C_α traces showing open (holo and L-malate bound structures; salmon) and closed (DHAP, itaconate, maleate, and citraconate bound structures; light green) states of PhNadA. (B) Close-up of holo and DHAP bound structures indicating side chain clashes.

Figure 3.

(A) Stereo diagram of the superimposition of DHAP bound to the [4Fe-4S] cluster in PhNadA onto PGH bound to zinc (cyan) in FucA (green). (B) Superimposition of the [4Fe-4S] cluster with bound DHAP onto the [4Fe-4S] cluster in the itaconate bound structure of PhNadA. (C) Stereo diagram (left) and schematic drawing (right) of intermediate 5 based on the result in panel B, with C₁ and C_β joined, followed by energy minimization with positional restraints applied to the protein atoms.