Newly identified essential amino acid residues affecting Δ8-sphingolipid desaturase activity revealed by site-directed mutagenesis

In order to identify amino acid residues crucial for the enzymatic activity of Δ8-sphingolipid desaturases, a sequence comparison was performed among Δ8-sphingolipid desaturases and Δ6-fatty acid desaturases 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 Δ8-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, D210, 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 Δ8-sphingolipid desaturase activity and aids in our understanding of the structure–function relationships of the membrane-bound desaturases.

► Fifteen conserved amino acid residues in Δ8-sphingolipid desaturase were identified.
► Ten of them are significantly important for the desaturase activity.
► Five of the ten are crucial for the Δ8-sphingolipid desaturase activity.
► Three of the ten markedly affect the ratio between the products of the desaturase.