Conformational Studies on the Δ8(E,Z)-Sphingolipid Desaturase from Helianthus annuus with Chiral Fluoropalmitic Acids As Mechanistic Probes

The Delta(8)-sphingolipid desaturase from sunflower (Helianthus annuus) converts phytosphinganine into a mixture of Delta(8)-(E)- and -(Z)-phytosphingenines by removal of two syn-hydrogen atoms from anti-, and gauche-conformations of the substrate. With chiral (R)-6-, (S)-6-, (R)-7-, and (S)-7-fluoropalmitic acids the importance of conformations for the formation of (E)- and (Z)-isomers was investigated by using growing yeast cells expressing the desaturase from H. annuus. The fluoropalmitic acids were readily incorporated into a series of fluorinated phytosphinganines. The desaturation products of the major C-18-fluorophytosphinganine demonstrate that different conformations of the relevant aliphatic segment of the sphingolipids can be exposed to the active center of the enzyme resulting in (E)- or (Z)-fluoroalkenes. The presence of a fluorine atom at the position of the initial hydrogen removal C8-H-R led to a complete suppression of the desaturation reaction, while replacement of C8-H-S with fluorine generated a mixture of mainly (Z)- and trace amounts of (E)-fluoroolefine. Fluorine at C9 of the phytosphinganine precursors did not interfere with the initial C-H activation step and produced (E)- and (Z)-fluoroalkenes in the same ratio as observed for the nonfluorinated precursors. Hydroxylated byproducts of the desaturation process were not observed. These results strongly support the importance of conformations of the transition states during desaturation as the relevant criterion for the relative ratio of (E)- and (Z)-alkenes.