Prediction of the active-site structure and NAD(+) binding in SQD1, a protein essential for sulfolipid biosynthesis in Arabidopsis

Sulfolipids of photosynthetic bacteria and plants are characterized by thei r unique sulfoquinovose headgroup, a derivative of glucose in which the 6-h ydroxyl group is replaced by a sulfonate group. These sulfolipids have been discussed as promising anti-tumor and anti-HIV therapeutics based on their inhibition of DNA polymerase and reverse transcriptase. To study sulfolipi d biosynthesis, in particular the formation of UDP-sulfoquinovose, we have combined computational modeling with biochemical methods. A database search was performed employing the derived amino acid sequence from SQD1, a gene involved in sulfolipid biosynthesis of Arabidopsis thaliana. This sequence shows high similarity to other sulfolipid biosynthetic proteins of differen t organisms and also to sugar nucleotide modifying enzymes, including UDP-g lucose epimerase and dTDP-glucose dehydratase. Additional biochemical data on the purified SQD1 protein suggest that it is involved in the formation o f UDP-sulfoquinovose, the first step of sulfolipid biosynthesis. To underst and which aspects of epimerase catalysis may be shared by SQD1, we built a three-dimensional model of SQD1 using the 1.8 Angstrom crystallographic str ucture of UDP-glucose 4-epimerase as a template. This model predicted an NA D(+) binding site, and the binding of NAD(+) was subsequently confirmed by enzymatic assay and mass spectrometry. The active-site interactions togethe r with biochemical data provide the basis for proposing a reaction mechanis m for UDP-sulfoquinovose formation. (C) 1999 Academic Press.