A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

Abstract

Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-⌬12 desaturase and a processive bifunctional oleoyl-⌬12/linoleoyl-3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional ⌬12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional ⌬12/3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model.

Caenorhabditis elegans Δ12-Desaturase FAT-2 Is a Bifunctional Desaturase Able to Desaturate a Diverse Range of Fatty Acid Substrates at the Δ12 and Δ15 Positions

“…When expressed in yeast cells, CeFAT-2 displays characteristics of a bifunctional ⌬12/⌬15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to desaturate fatty acids ranging from C14 to C18. Finally, phylogenetic analysis suggests that despite possessing a bifunctional ⌬12/⌬15 desaturation activity, CeFAT-2 appears to have arisen independently from other reported dual ⌬12/⌬15-desaturases from fungi (7,8) and protozoa (9).…”

“…These modes of regioselectivity are not mutually exclusive as illustrated by several bifunctional fungal ⌬12/3-desaturases. For example, in addition to the ⌬12-fatty acid desaturase members, separate bifunctional ⌬12/3-fatty acid desaturases have been identified from fungi Fusarium moniliforme (7) and Aspergillus nidulans (8). These bifunctional enzymes desaturate oleic acid C18:1…”

“…Additionally, these fungal desaturases also convert 6 substrates, including LA, ␥-linolenic acid (C18:3 ⌬6,9,12 ), dihomo-␥-linolenic acid (C20:3 ⌬8,11,14 ), and arachidonic acid (C20:4 ⌬5, 8,11,14 ) to their 3 products and show an obvious preference for the C18 substrates, especially LA (7,8). A bifunctional ⌬12/⌬15-desaturase that directs the synthesis of highly unusual hexadecatrienoic acid (C16:3 ⌬6,9,12 , 1) has also been isolated from the free-living soil protozoon Acanthamoeba cas-tellanii (9) and phytopathogenic fungus, Claviceps purpurea (10).…”

“…The identity of C16:1 ⌬7 and C16: 2 ⌬7,10 peaks is confirmed by MS analysis on both FAMEs and DMOX adducts of the FAMEs which display mass spectra similar to the standard C16:1 ⌬7 and C16:2 ⌬7,10 (data not shown). C20:5 ⌬5,8,11,14,17 (3) rather than C18:3 ⌬9,12,15 is a major polyunsaturated fatty acid in C. elegans, thus it is of interest to know whether CeFAT-2 contributes to the C20:5 ⌬5, 8,11,14,17 production. CeFAT-1 is the 3-desaturase converting 6 fatty acid C20:4 ⌬5,8,11,14 to 3 fatty acid C20:5 ⌬5, 8,11,14,17 .…”

“…C20:5 ⌬5,8,11,14,17 (3) rather than C18:3 ⌬9,12,15 is a major polyunsaturated fatty acid in C. elegans, thus it is of interest to know whether CeFAT-2 contributes to the C20:5 ⌬5, 8,11,14,17 production. CeFAT-1 is the 3-desaturase converting 6 fatty acid C20:4 ⌬5,8,11,14 to 3 fatty acid C20:5 ⌬5, 8,11,14,17 . We show in yeast cells that CeFAT-1 is able to produce C20:5 ⌬5, 8,11,14,17 from fed C20:4 ⌬5,8,11,14 at a conversion rate of 18%, but only a trace amount (0.44%) of C20:5 ⌬5, 8,11,14,17 can be produced by CeFAT-2 at conversion rate of 2% (supplemental Table S2).…”