Crystal structure of SQD1, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose

Abstract

The SQD1 enzyme is believed to be involved in the biosynthesis of the sulfoquinovosyl headgroup of plant sulfolipids, catalyzing the transfer of SO(3)(-) to UDP-glucose. We have determined the structure of the complex of SQD1 from Arabidopsis thaliana with NAD(+) and the putative substrate UDP-glucose at 1.6-A resolution. Both bound ligands are completely buried within the binding cleft, along with an internal solvent cavity which is the likely binding site for the, as yet, unidentified sulfur-donor substrate. SQD1 is a member of the short-chain dehydrogenase/reductase (SDR) family of enzymes, and its structure shows a conservation of the SDR catalytic residues. Among several highly conserved catalytic residues, Thr-145 forms unusually short hydrogen bonds with both susceptible hydroxyls of UDP-glucose. A His side chain may also be catalytically important in the sulfonation.

Figure 1

A scheme of the SQD1 reaction mechanism derived from Essigmann et al. (9).

Figure 2

(A) Ribbon drawing of the SQD1 structure shown in stereoview. The large domain is shown in blue with additional 7th strand of Rossmann motif in green; small domain is gold; the C terminus is labeled C. Bound NAD (aqua) and UDP-glucose (red) are shown as space-filling models. Asterisk indicates β-hairpin insertion loop. (B) View of SQD1 homodimer along C2 dimer axis showing interaction of four-helix bundle. The figure was generated by using molscript (46) and raster3d (47).

Figure 3

(A) View of the buried ligands (NAD⁺ in yellow and UDP-glucose in red) and water molecules (blue) within the protein (SS is the presumptive sulfur donor site). The C-terminal flap, which may open to allow access to the sugar-dinucleotide-binding site, is shown in gold. (B) Surface representation highlighting the water molecules (blue spheres) at entrances of the large (LC) and small (SC) solvent channels leading into the SQD1 binding cleft.

Figure 4

Schematic diagram of potential hydrogen-bonding interactions of bound NAD⁺ and UDP-glucose with the protein. All potential interactions (dashed lines) are within 3.2 Å except those involving Asn-119 N^δ (3.4 Å) and Arg-36 N^ɛ (3.4 Å). Circles represent bound water molecules; W1 and W2 are waters Wat1 and Wat2, respectively.

Figure 5

The active center of SQD1. (A) Stereoview showing the bound NAD⁺ (aqua), UDP-glucose (red), and Wat2 (arrowhead) with respect to presumed catalytic residues of SQD1 active site. (B) A close-up view of the tightly bound water Wat2 and its interactions with Thr-145 and the hexose ring. All hydrogen bonds (dotted lines) involving Thr-145 and bound water are 2.4 Å. The figure was generated by using setor (48).