Identification of amino acids conferring chain length substrate specificities on fatty alcohol-forming reductases FAR5 and FAR8 from Arabidopsis thaliana

Abstract

Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.

Keywords: Enzyme Structure; Fatty Acyl Reductase; Fatty Alcohol; Lipid Metabolism; Plant Biochemistry; Protein Engineering; Site-directed Mutagenesis.

FIGURE 1.

FAR structural domains and protein sequence alignment of Arabidopsis FAR5 and FAR8. A, schematic of the structural domains of FAR proteins. FAR enzymes, minus possible subcellular localization signals (e.g. plastid targeting), are ∼500 amino acids in length and contain an NAD(P)H binding Rossmann-fold domain (light gray) and a FAR_C domain (dark gray) at the N and C termini, respectively. The GXXGXX(G/A) predicted NADPH-binding motif as well as the predicted active site motif YXXXK are indicated (where X represents any amino acid). B, protein sequence alignments of FAR5 and FAR8, which are 85% similar at the amino acid level. Identical amino acids are highlighted in black, and physiochemically similar amino acids are highlighted in gray. Arrows indicate domain swap sites, and asterisks indicate site-specific mutagenesis sites.

FIGURE 2.

Gas chromatograms of internal lipids of yeast expressing Arabidopsis FAR5, FAR8, or FAR8-S363P. The empty vector pYES2-His₆/T7 tag acted as a negative control. Transformants were cultured in galactose media to induce protein expression. Fatty acids were transmethylated to their corresponding methyl esters, and fatty alcohol hydroxyl groups were derivatized to trimethylsilyl ethers before separation by GC and detection by flame ionization. The peaks corresponding to pentadecanol (15:0-OH) internal standard (IS), 16:0-OH and 18:0-OH are indicated, as well as saturated and monounsaturated C16 and C18 fatty acids (FA).

FIGURE 3.

Amino acids important for FAR5 and FAR8 enzyme stability and activity. A, left, schematics of FAR5 and FAR8 variants. The FAR5 variants are in black, and FAR8 variants are in gray, with positions of site-specific mutations indicated above each protein schematic. Right, the amounts of nonsecreted fatty alcohols produced by yeast-expressing FARs, where values are expressed in μg/A₆₀₀ unit ± S.D. (n = 4). B, protein levels of FAR5 and FAR8 variants expressed in yeast. Western blots (top) were performed using anti-T7 mouse antibody to detect the N-terminal T7 epitope in the protein fusions. The Coomassie-stained gel is shown at the bottom to indicate equal loading. The positions of the protein size markers (in kDa) are indicated to the left of the Western blot and stained gel.

FIGURE 4.

Amounts of total fatty alcohols produced by FAR5 and FAR8 variants expressed in yeast. A, internal fatty alcohol content of yeast cells (nonsecreted fatty alcohols). B, fatty alcohols found in supernatant of yeast cultures (secreted fatty alcohols). C, total fatty alcohols produced by yeast expressing a FAR variant (combined nonsecreted and secreted fatty alcohol content). Values are expressed in μg/A₆₀₀ unit ± S.D. (n = 4). Yeast cultures were grown for 48 h in galactose media for protein induction before lipid extraction and analysis.

FIGURE 5.

Domain swaps between FAR5 and FAR8. A, left, schematics of FAR5 and FAR8 domain swap chimeras, with the portions from FAR5 in black and the portions from FAR8 in gray. The active FAR8-S363P mutant, denoted as FAR8R, was used in all domain swaps. Right, amounts of nonsecreted fatty alcohols produced by yeast expressing FAR5, FAR8R, or a FAR5/FAR8 chimera, where values are expressed in μg/A₆₀₀ unit ± S.D. (n = 4). B, protein levels of FAR5, FAR8R, and FAR5/FAR8 chimeras expressed in yeast. Western blots (top) and Coomassie-stained gel (bottom) are as described in Fig. 3 legend.

FIGURE 6.

Reciprocal amino acid substitutions in FAR5 and FAR8. A, protein alignment of amino acid residues 345–388 of FAR5 and FAR8. Identical amino acids are highlighted in black, and physiochemically similar amino acids are highlighted in gray. Asterisks indicate the four amino acids (347, 355, 363, and 377) targeted for analysis in this region by site-specific mutagenesis. B, analysis of amino acids 355 and 377 affecting substrate specificity of FAR5. C, analysis of amino acids 355 and 377 affecting substrate specificity of FAR8. The S363P mutation in FAR8, denoted as FAR8R, was present in all FAR8 variants. The top parts of B and C are graphs reporting the amounts of nonsecreted fatty alcohols produced by FAR5 and FAR8 variants, where values are expressed in μg/A₆₀₀ unit ± S.D. (n = 4). The bottom parts of B and C report the protein levels of FAR5 and FAR8 variants. Western blots (top) and Coomassie-stained gel (bottom) are as described in Fig. 3 legend.

FIGURE 7.

FAR in vitro assays with yeast microsomes containing FAR5 and FAR8 variants. A, representative experiment showing separation of lipids recovered from in vitro FAR assays by TLC. Assays were conducted in the presence of ¹⁴C-radiolabeled 18:0-CoA (top) or ¹⁴C-radiolabeled 16:0-CoA (bottom), and microsomes were extracted from yeast expressing the FAR indicated below the plate images. The radiolabeled fatty alcohols were identified by co-migration with unlabeled standards. B, protein levels of FAR5 and FAR8 variants in yeast microsomes. Western blots (top) and Coomassie-stained gel (bottom) are as described in Fig. 3 legend.