A cotton PATE cDNA clone has a 1.7-kb insert with an coding region for 410 amino acids, lacking codons for the three N-terminal amino acids. The predicted amino acid sequence of the PATE preprotein has a characteristic stromal-targeting domain and a 63% identity to the Arabidopsis FatB1 thioesterase sequence. A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-ACP thioesterase (FatB1) gene. The gene spans 3.6 kb with six exons and five introns. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein is identified as a FatB thioesterase from its deduced amino acid sequence similarity to those of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential promoter/enhancer elements including basic helix-loop-helix elements (E box). Alkaline blot hybridization of cotton genomic DNA suggests the presence at least two FatB1 thioesterase genes in cotton. Four plasmid constructs for both constitutive and seed-specific anti-sense RNA suppression and gene-transgene co- suppression of PATE gene expression were successfully generated. Two overlapping cotton genomic clones were found to encompass a Δ-12 fatty acid desaturase (FAD2-3) gene. The continuous FAD2-3 coding region is 1,155 bp and would encode a protein of 384 amino acids. The FAD2-3 gene has one large intron of 2,967 bp entirely within its 5'-untranslated region. Several potential promoter/enhancer elements, including several light responsive motifs occur in the 5'-flanking region. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.

Polyunsaturated fatty acids are major structural components of plant chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases (designated FAD2 and FAD3) desaturate 75% of the fatty acids in the endoplasmic reticulum. The w -6 fatty acid desaturase (FAD2) may be responsible for cold acclimation response, since polyunsaturated phospholipids are important in helping maintain plant viability at lowered temperatures. To study regulation of FAD2 gene expression in cotton, a FAD2 gene was isolated from two genomic libraries using an Arabidopsis FAD2 hybridization probe and a cotton FAD2 5¢ -flanking region gene-specific probe, respectively. A cotton FAD2 gene was found to be in two overlapping genomic clones by physical mapping and DNA sequencing. The cloned DNA fragments are identical in size to cotton FAD2 genomic DNA fragments shown by genomic blot hybridization. The cotton FAD2 coding region has 1,155 bp with no introns and would encode a putative polypeptide of 384 amino acids. The cotton FAD2 enzyme has a high identity of 75% with other plant FAD2 enzymes. The enzyme has three histidine-rich motifs that are conserved in all plant membrane desaturases. These histidine boxes may be the iron-binding domains for reduction of oxygen during desaturation. To confirm that this FAD2 enzyme is functional, a plasmid construct containing the cotton FAD2 coding region was transformed into Saccharomyces cerevisiae. The transformed yeast cells were able to catalyze the conversion of oleic acid (C18:1) into linoleic acid (C18:2). The FAD2 gene contains an intron of 2,967 bp in its 5¢ -flanking region, 11 bp upstream from the initiation codon. The intron could be essential for transcriptional regulation of FAD2 gene expression. Several putative promoter elements occur in the 5¢ -flanking region of this gene. A potential TATA basal promoter element occurs at 41 bp upstream from the cap site. Two presumptive helix-loop-helix (bHLH) motifs that may be seed-specific promoter elements are located at 109 bp and 135 bp upstream from the potential cap site.

This study provides insight into the structure/function relationship between desaturases and acetylenases, and indicates amino acid residues within acetylenases which influence reaction outcome. <i>Oleate desaturases</i> belong to a family of enzymes capable of introducing cis double bonds between C12 - C13 in oleate esters. Acetylenases are a subset of oleate desaturase enzymes which introduce a triple bond in the C12 - C13 position of linoleate. To better understand which amino acids could be responsible for differentiating the activity of acetylenases from typical desaturases, a total of 50 protein sequences were used to compare the two classes of enzymes resulting in the identification of 11 amino acid residues which are conserved within either separate family but differ between the two groups of enzymes. These identified amino acid residues were then singularly altered by site-directed mutagenesis to test their role in fatty acid modification. Specifically, the wild type acetylenase, Crep1 from <i>Crepis alpina</i>, and a number of point mutants have been expressed in <i>Saccharomyces cerevisiae</i>, followed by fatty acid analysis of the resulting cultures. Results indicate the importance of 4 amino acid residues within Crep1 (Y150, F259, H266, and V304) with regards to desaturase and acetylenase chemoselectivity, stereoselectivity, and/or substrate recognition. The F259L mutation affected the acetylenase by converting it to an atypical FAD2 capable of producing both cis and trans isomers. The V304I mutation resulted in the conversion of Crep1 into a stereoselective FAD2, where only the cis isomers of 16:2 and 18:2 were produced. The Y150F mutation led to a loss of acetylenase activity without affecting the inherent desaturase activity of Crep1. The H266Q mutation appears to affect substrate selection causing an inability to bind substrate (16:1-9c and/or 18:1-9c) in a cisoid conformation, resulting in an increased accumulation of trans product. The changes in enzyme activity detected in cultures expressing Crep1 mutants demonstrate the profound effect that exchanging as little as one amino acid can have on an enzyme properties. Enzymes retain some conservation of amino acids necessary for activity, such as those involved in metal ion binding, whereas subtle changes can affect overall enzyme function and catalysis.

This study provides insight into the structure/function relationship between desaturases and acetylenases, and indicates amino acid residues within acetylenases which influence reaction outcome. <i>Oleate desaturases</i> belong to a family of enzymes capable of introducing cis double bonds between C12 - C13 in oleate esters. Acetylenases are a subset of oleate desaturase enzymes which introduce a triple bond in the C12 - C13 position of linoleate. To better understand which amino acids could be responsible for differentiating the activity of acetylenases from typical desaturases, a total of 50 protein sequences were used to compare the two classes of enzymes resulting in the identification of 11 amino acid residues which are conserved within either separate family but differ between the two groups of enzymes. These identified amino acid residues were then singularly altered by site-directed mutagenesis to test their role in fatty acid modification. Specifically, the wild type acetylenase, Crep1 from <i>Crepis alpina</i>, and a number of point mutants have been expressed in <i>Saccharomyces cerevisiae</i>, followed by fatty acid analysis of the resulting cultures. Results indicate the importance of 4 amino acid residues within Crep1 (Y150, F259, H266, and V304) with regards to desaturase and acetylenase chemoselectivity, stereoselectivity, and/or substrate recognition. The F259L mutation affected the acetylenase by converting it to an atypical FAD2 capable of producing both cis and trans isomers. The V304I mutation resulted in the conversion of Crep1 into a stereoselective FAD2, where only the cis isomers of 16:2 and 18:2 were produced. The Y150F mutation led to a loss of acetylenase activity without affecting the inherent desaturase activity of Crep1. The H266Q mutation appears to affect substrate selection causing an inability to bind substrate (16:1-9c and/or 18:1-9c) in a cisoid conformation, resulting in an increased accumulation of trans product. The changes in enzyme activity detected in cultures expressing Crep1 mutants demonstrate the profound effect that exchanging as little as one amino acid can have on an enzyme properties. Enzymes retain some conservation of amino acids necessary for activity, such as those involved in metal ion binding, whereas subtle changes can affect overall enzyme function and catalysis.