Mechanistic and structural insights into the regioselectivity of an acyl-CoA fatty acid desaturase via directed molecular evolution

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.

FIGURE 1.

Complementation assay of the wild type cricket Δ9-desaturase (AdD9des), wild type cricket Δ12/Δ9-desaturase (AdD12/9des), and cricket Δ12/Δ9-desaturase mutants 1.1, 2.1, 2.2, and 2.3 in ole1Δ. A dilution series of each sample was spotted on SC-Ura agar plates containing galactose (left) or glucose (right). Yeast cells transformed with the pYES2 vector served as a negative control. OD, optical density.

FIGURE 2.

Amino acid substitutions identified in 3 selected random mutants of the cricket Δ12/Δ9-desaturase. A, proposed topology model of the cricket acyl-CoA Δ12/Δ9 fatty acid desaturase. The positions of selected random mutations are indicated by black circles. ER lumen, endoplasmic reticulum lumen. B, partial ClustalW alignment of the cricket Δ9-desaturase (AdD9des), cricket Δ12/Δ9-desaturase (AdD12/9des), and cricket Δ12/Δ9-desaturase mutants 1.1, 2.1, 2.2 and 2.3. Identical residues are shaded, conserved histidine clusters 1 and 2 are underlined, and asterisks below the alignment indicate selected random mutations.

FIGURE 3.

Regioselectivity of wild type (AdD12/9des) and mutant cricket Δ12/Δ9-desaturases. 16:0 and 18:1 conversion efficiencies (%) were calculated from relative substrate and product levels (percentage of total fatty acids) as 100 × product/(substrate + product). Data represent means ± S.D. of three replicate cultures.

FIGURE 4.

A–D, GC analysis of ole1Δ cells expressing the wild type cricket Δ12/Δ9-desaturase (A and C) or desaturase mutant 2.2 (B and D) when supplied with 17:1^Δ10 (A and B) or 18:1^Δ9 (C and D). FID, flame ionization detector. E, mass spectrum of the 4,4-dimethyloxazoline derivative of the 14:1^Δ9 peak in ole1Δ cells expressing the cricket desaturase mutant 2.2. A gap of 12 mass units between the ions m/z = 196 and 208 is diagnostic for a double bond at position Δ9.

FIGURE 5.

Δ9-desaturation activity of wild type (AdD9des) and mutant cricket Δ9-desaturases. 16:0 and 18:0 conversion efficiencies (%) were calculated from relative substrate and product levels (percentage of total fatty acids) as 100 × product/(substrate + product). Data represent means ± S.D. of three replicate cultures.

FIGURE 6.

Regioselectivity of the T. castaneum Δ12-desaturase (TcD12des), A. thaliana FAD2 Δ12-desaturase (AtD12des), wild type cricket Δ12/Δ9-desaturase (AdD12/9des), and targeted and random mutants of the cricket Δ12/Δ9-desaturase. 16:0 and 18:1 conversion efficiencies (%) were calculated from relative substrate and product levels (percentage of total fatty acids) as 100 × product/(substrate + product). 16:0/18:1 conversion ratios are shown relative to the wild type AdD12/9des desaturase. Data represent means ± S.D. of three replicate cultures.