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 ⌬x regioselectivity. After two selection rounds, variants of the bifunctional ⌬12/⌬9-desaturase from the house cricket (Acheta domesticus) exhibited increased ⌬9-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 ⌬9-desaturation of shorter chain acyl substrates and allowed for the redesign of the cricket ⌬12/⌬9-desaturase into a 16:0-specific ⌬9-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 ⌬12-desaturation.Fatty acid desaturases are nonheme diiron-containing enzymes that introduce double bonds within fatty acyl chains and belong to two main groups. The soluble desaturases are localized in the stroma of plastids where they typically introduce the first double bond in saturated fatty acids esterified to an acyl carrier protein. In contrast, the majority of the fatty acid desaturases are integral membrane proteins recognizing acyl chains esterified to either CoA or phospholipids. Although a similar diiron active site is present in membrane-bound desaturases, they are unrelated to soluble desaturases and are believed to have evolved independently (1). Although crystal structures for soluble desaturases from both castor (Ricinus communis) and the English Ivy (Hedera helix) (2, 3) have paved the way for engineered variants displaying intriguing novel catalytic activities and specificities (4 -6), our understanding of the structure-function relationship of membrane-bound desaturases remains limited and scattered at best. Consequently, engineering of this class of desaturase enzymes has lagged behind that of the soluble desaturases.Historically, three main types of regioselectivity have been distinguished (7,8). Desaturases with ⌬x regioselectivity introduce the first double bond x carbon atoms away from the carboxyl end. A second group comprises y-desaturases that insert a double bond at position y referenced from the methyl end. Finally, vϩz-desaturases require a preexisting double bond as a reference point (v) and generate the new double bond z carbon atoms further along the acyl chain. These modes of r...

Trans-vaccenate is Δ13-desaturated by FADS3 in rodents

“…Rat hepatocytes were incubated for 24 h with media supplemented with TVA or [1][2][3][4][5][6][7][8][9][10][11][12][13] C]TVA and with a control medium without FA. RA (R t = 27.5 min) produced by ⌬ 9-desaturation of TVA was easily detected (7.6 ± 3.2% of FA, n = 5) in the cells incubated with TVA or 13 C-TVA ( Fig.…”

“…The pure trans 11, trans 13-18:2 was obtained from Larodan (Malmö, Sweden). [1][2][3][4][5][6][7][8][9][10][11][12][13] C] trans -vaccenic acid (chemical purity 95%, isotopic enrichment 99%) was synthesized by the Commissariat à l'Energie Atomique (CEA, Gif-sur-Yvette, France) according to well-established procedures ( 27,28 ). FA albuminic complexes were prepared as described ( 26 ).…”

Biotechnological potential of insect fatty acid-modifying enzymes

“…The mFADs and their mutants are typically functionally characterized in the yeast S. cerevisiae [151] or in baculovirus-insect cell expression systems [79]. These experiments led to the identification of sequence determinants of both acyl-CoA and acyl-lipid mFAD specificities in diverse organisms, such as transmembrane helices or conserved histidine-rich motifs [55,59,[152][153][154][155][156][157][158][159][160][161][162]. Buček et al [82] identified a critical amino acid residue in the transmembrane domain of an mFAD from Manduca sexta (MsexD3) that determines the specificity and ability of this desaturase to catalyze biosynthesis of FAs containing three conjugated double bonds via E/Z14 desaturation from diunsaturated FAs.…”

Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases

“…Site-directed mutagenesis based on amino acid sequence comparison has been used to identify amino acids participating in the substrate specifi city of Mucor rouxii D6d ( 15 ), Siganus canaliculatus ⌬ 4 and D5d/D6d ( 16 ), and marine copepod ⌬ 9 desaturase ( 17 ). The regioselectivity of house cricket ⌬ 12/ ⌬ 9 desaturase was investigated using chemical mutagenesis and yeast complementation assays ( 18 ). Moreover, fatty acid-modifying enzymes with protein structures similar to, but chemoselectivities different from, the fatty acid desaturases have been used to swap the function of Arabidopsis oleate 12-desaturase and hydroxylase ( 19 ) and to alter the product partitioning between Crepis alpina ⌬ 12 desaturase and acetylenase ( 20 ) and Momordica conjugase itself ( 21 ).…”