Native uridine 5'-diphosphate-sulfoquinovose synthase,SQD1, from spinach purifies as a 250-kDa complex

Sulfoquinovosyldiacylglycerol is a polar lipid present in photosynthetic membranes. It contributes to the negative surface charge of the membrane and plays a pivotal role under phosphate stress. The SQD1 protein is the key enzyme involved in the formation of the sulfolipid head group precursor, uridine 5(')-diphosphate (UDP)-sulfoquinovose, from UDP-glucose and sulfite. A cDNA encoding the spinach SQD1 protein was isolated and functionally expressed in Escherichia coli. The recombinant enzyme was compared to the native enzyme purified from isolated spinach chloroplasts. While the K(m) for UDP-glucose was indistinguishable for the two forms, the K(m) for sulfite was more than fourfold lower (< microM) for the native enzyme. Sizing by gel filtration indicated that the native form purified as a large complex of approximately 250 kDa, which is more than twice as large as the calculated size for the homodimer. It is proposed that in vivo SQD1 forms a complex with accessory proteins.     

Questions remaining in sulfolipid biosynthesis: a historical perspective

The plant sulfolipid sulfoquinovosyldiacylglycerol was discovered by A.A. Benson in the late 1950s. The increasing availability of radioisotope-containing biological substrates such as ³⁵S-sulfate provided the means to discover novel biological compounds and to sketch out their biosynthetic pathways. During this time the structure of sulfolipid with its 6-deoxy-6-sulfo-α-d-glucose (sulfoquinovose) headgroup was determined. Immediately, the origin of this unusual biological sulfonic acid mystified the scientific community and several proposals for its biosynthesis were developed and tested. Strong supportive evidence for the nucleotide pathway of sulfolipid biosynthesis became available with the discovery of the bacterial and plant genes encoding the enzymes of sulfolipid biosynthesis during the 1990s. This latter work was based on the foundations laid by A.A. Benson and confirmed one initial hypothesis on sulfolipid biosynthesis. An abbreviated summary of the turning points in defining the mechanism for sulfolipid biosynthesis and remaining issues in sulfolipid biochemistry are provided.     

Identification of a gene for UDP-sulfoquinovose synthase of a green alga, Chlamydomonas reinhardtii, and its phylogeny.

Sulfoquinovosyl diacylglycerol is responsible for the structural and functional integrity of the photosystem II complex of a green alga, Chlamydomonas reinhardtii. We cloned a cDNA of C. reinhardtii containing an open reading frame for a protein 36-64% identical in the primary structure to known UDP-sulfoquinovose synthases, which are required for SQDG synthesis, in other organisms. Through the introduction of the cDNA, a cyanobacterial disruptant as to the UDP-sulfoquinovose synthase gene recovered the ability to synthesize sulfoquinovosyl diacylglycerol, thus confirming that the cDNA encodes the UDP-sulfoquinovose synthase. On the genome, the cDNA was divided into 14 exons, and the gene designated as SQD1 was present as one copy. The molecular phylogenetic tree for the UDP-sulfoquinovose synthase showed grouping of C. reinhardtii together with species that require sulfoquinovosyl diacylglycerol for the functioning of the PSII complex, but not with those that do not utilize the lipid for photosynthesis. The role of sulfoquinovosyl diacylglycerol in the functioning of the photosynthetic membranes might evolve in harmony with the system of the membrane lipid synthesis such as UDP-sulfoquinovose synthase gene.     

Ferredoxin-dependent glutamate synthase moonlights in plant sulfolipid biosynthesis by forming a complex with SQD1

UDP-sulfoquinovose synthase, SQD1, catalyzes the transfer of sulfite to UDP-glucose giving rise to UDP-sulfoquinovose, which is the head group donor for the biosynthesis of the plant sulfolipid sulfoquinovosyldiacylglyerol. The native SQD1 enzyme of spinach exists as a 250 kDa heteroprotein complex with much higher affinity for the substrate sulfite than the recombinant SQD1 protein itself. The SQD1 protein co-purified with nine proteins. Likely binding partners included rubisco activase, HSP70, and ferredoxin-dependent glutamate synthase (FdGOGAT). While the first two proteins are known to interact with many other proteins, the identification of FdGOGAT was most intriguing because this 160kDa protein contains an FMN cofactor known to bind sulfite in vitro. Using different constructs expressing recombinant forms of the multidomain protein FdGOGAT, it was demonstrated that the FMN-binding domain of FdGOGAT is essential for specific binding of the protein to SQD1. A model suggests that FdGOGAT could channel sulfite to SQD1.