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Title: Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

Author
item LI, S - Chinese Academy Of Sciences
item SONG, L - Chinese Academy Of Sciences
item YIN, W - Chinese Academy Of Sciences
item CHEN, Y - Chinese Academy Of Sciences
item Wang, Richard
item HU, Z - Chinese Academy Of Sciences
item ZHANG, G - Chinese Academy Of Sciences

Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2011
Publication Date: 12/20/2011
Citation: Li, S.F., Song, L.Y., Yin, W.B., Chen, Y.H., Wang, R., Hu, Z.M., Zhang, G.J. 2011. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis. Biochem Biophys Res Comm. 416:165-171.

Interpretive Summary: Fatty acid desaturases are a class of enzymes that introduce double bonds into fatty acyl chains to produce unsaturated and polyunsaturated fatty acids. These desaturases mainly belong to two distinct families, one soluble and the other membrane-bound. Compared with the soluble desaturases, the membrane-bound desaturases are more difficult to study, thus, there is little information about the structure-function relationships of the membrane-bound desaturases. ^8-sphingolipid desaturases are ubiquitous in plants and highly related to ^6-fatty acid desaturases that only exist in several plants to produce v-linolenic acids (GLA) and octadecatetraenoic acids (OTA). ^8-unsaturated sphingolipids were correlated with plants' tolerance to abiotic stresses such as chilling and aluminum. There is still little information about the effect of other residues on the enzyme activity of the membrane-bound desaturase besides the cytb5 HPGG motif and histidine box motif. We used site-directed mutagenesis to replace individual residues of a new ^8-sphingolipid desaturase gene BrD8A from Brassica rapa and then used yeast (Saccharomyces cerevisae) system to assess the enzyme activity of the constructed mutants. We identified some amino acid residues that are essential for the enzyme activity, and several residues that are important not only for the enzyme activity but also for the ratio of the two enzyme products, 8(Z)-C18- and 8(E)-C18-phytosphingenines.

Technical Abstract: In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated ^8-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D2l0, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the ^8-sphingolipid desaturase and aid in our understanding of the structure-function relationships of the membrane-bound desaturases.