Abstract
Membrane-bound fatty acid desaturases and related enzymes play a pivotal role in the biosynthesis of unsaturated and various unusual fatty acids. Structural insights into the remarkable catalytic diversity and wide range of substrate specificities of this class of enzymes remain limited due to the lack of a crystal structure. To investigate the structural basis of the double bond positioning (regioselectivity) of the desaturation reaction in more detail, we relied on a combination of directed evolution in vitro and a powerful yeast complementation assay to screen for Δx regioselectivity. After two selection rounds, variants of the bifunctional Δ12/Δ9-desaturase from the house cricket (Acheta domesticus) exhibited increased Δ9-desaturation activity on shorter chain fatty acids. This change in specificity was the result of as few as three mutations, some of them near the putative active site. Subsequent analysis of individual substitutions revealed an important role of residue Phe-52 in facilitating Δ9-desaturation of shorter chain acyl substrates and allowed for the redesign of the cricket Δ12/Δ9-desaturase into a 16:0-specific Δ9-desaturase. Our results demonstrate that a minimal number of mutations can have a profound impact on the regioselectivity of acyl-CoA fatty acid desaturases and include the first biochemical data supporting the acyl-CoA acyl carrier specificity of a desaturase able to carry out Δ12-desaturation.
Highlights
BankTM/EBI Data Bank with accession number(s) AF338465 and EU159448. 1 To whom correspondence should be addressed: CSIRO Plant Industry, P
Membrane-bound fatty acid desaturases have been the subject of only a limited number of regioselectivity studies
In an attempt to further increase our understanding of the structural principles that determine the regioselectivity of membrane-bound fatty acid desaturases, we relied on directed evolution in vitro rather than on more rational protein design principles that formed the basis of previous studies
Summary
Yeast Strains and Expression Vectors—Saccharomyces cerevisiae strains used for the expression of wild type and mutant desaturase genes included S288C (Mat␣, SUC2, gal, mal, mel, flo, flo, hap1) and an ole deletion mutant (his3⌬1, leu2⌬0, ura3⌬0, ole1⌬::kanMX4) (ATCC 4024422) [18], designated hereupon as ole1⌬. Precultures of ole1⌬ transformants were set up in 5 ml of SC-Ura medium supplemented with 2% glucose, 1% Tergitol, and any exogenous fatty acid required. After incubation overnight at 28 °C, yeast cells were washed with 5 ml of water and resuspended in 5 ml of SC-Ura induction medium containing 2% galactose, 1% Tergitol, and the appropriate fatty acid. Induction cultures were incubated for 3 days at 28 °C and washed with equal volumes of 1% Tergitol, 0.5% Tergitol, and water to remove any unincorporated fatty acids. Ole1⌬ Complementation Assay—The desaturase library was divided in 10-l aliquots, transformed into ole1⌬ using the yeast-1 transformation kit (Sigma-Aldrich), and plated on SCUra medium containing 2% glucose, 0.5 mM 16:1⌬9, and 1% Tergitol. After a second overnight incubation, cell pellets were washed with an equal volume of water and resuspended in 45 ml of SC-Ura-Trp induction medium containing 2% galactose. Statistical Analysis—One-way analysis of variance and pairwise two-tailed t tests (Holm-Bonferroni) were done with R-2.11.1 for Windows
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