Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity

Abstract

Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

Figure 1

Sequence alignments of the first 167 residues of the borage Δ⁶-desaturase (boofd6) with the Arabidopsis FAD2 (arthfad2) and FAD3 (arthfad3) microsomal desaturases, sunflower Δ⁸-sphingolipid desaturase (heand8), cyanobacteria Δ⁶ desaturase (synd6), and borage Cyt b₅ (boofb5). The conserved HPGG motif is underlined, with the essential His (H⁴¹) indicated by a star. The position of the internal Met residues (Met-113 and Met-147) are boxed and arrowed. The conserved His residues of the first “His box” of the microsomal desaturases are also indicated by black dots.

Figure 2

Northern-blot analysis of transgenic Arabidopsis plants expressing the full-length borage Δ⁶-desaturase (AT6) compared with lines expressing two N-terminally truncated forms (Δ112 and Δ146). The approximate size of the detected transcript is indicated. A control transformed Arabidopsis (WT) sample was also probed. Ten micrograms of RNA was loaded in each lane.

Figure 3

Northern-blot analysis of transgenic Arabidopsis plants expressing either the full-length borage Δ⁶-desaturase (AT6) or a mutated form in which His-41 was converted to an Ala (H41A). Approximately 10 μg of RNA was loaded for both samples, as judged by ethidium bromide staining (B), and the approximate size of the detected transcript is indicated (A).