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 their unique sulfoquinovose headgroup, a derivative of glucose in which the 6-hydroxyl 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 sulfolipid 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 different organisms and also to sugar nucleotide modifying enzymes, including UDP-glucose epimerase and dTDP-glucose dehydratase. Additional biochemical data on the purified SQD1 protein suggest that it is involved in the formation of UDP-sulfoquinovose, the first step of sulfolipid biosynthesis. To understand which aspects of epimerase catalysis may be shared by SQD1, we built a three-dimensional model of SQD1 using the 1.8 A crystallographic structure of UDP-glucose 4-epimerase as a template. This model predicted an NAD(+) binding site, and the binding of NAD(+) was subsequently confirmed by enzymatic assay and mass spectrometry. The active-site interactions together with biochemical data provide the basis for proposing a reaction mechanism for UDP-sulfoquinovose formation.
Essigmann
B
B
Department of Biochemistry, Michigan State University, East Lansing, Michigan, 48824-1319, USA.
Hespenheide
B M
BM
Kuhn
L A
LA
Benning
C
C
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D016208:Databases, Factual; D004926:Escherichia coli / Q000201:enzymology; D006836:Hydro-Lyases / Q000737:chemistry; D008055:Lipids / Q000096:biosynthesis*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014534:UDPglucose 4-Epimerase / Q000737:chemistry; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D008055:Lipids; D010940:Plant Proteins; C110849:SQD1 protein, Arabidopsis; C015518:sulfolipids; D009243:NAD; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase; D014534:UDPglucose 4-Epimerase; D014532:Uridine Diphosphate Glucose