Integrated structural, computational, and genetic analyses link KASII-A^L263F to increased palmitic acid composition...

Soybean seed oil quality is governed by the relative proportions of five major fatty acids. Most genetic studies of soybean fatty acid mutants have evaluated allelic effects primarily on fatty acid composition, rather than quantifying their corresponding contents. Because fatty acid composition is expressed as relative percentage of total fatty acids, an increase in the proportion of one fatty acid is necessarily accompanied by a relative decrease in others, making it difficult to distinguish compositional shifts from changes in the actual amount accumulated per unit seed mass. In this study, we characterized PE1604, an EMS-induced mutant derived from the Korean soybean cultivar Pungsannamul and evaluated fatty acid phenotypes using both composition and contents. Sequence analysis identified a C to T transition in KASII-A (Glyma.17G047000) that resulted in an L263F amino acid substitution at a leucine residue conserved among KASII-A homologs from 11 species of the family Fabaceae. AlphaFold2 models superposed on the Escherichia coli (E. coli) FabF complex with crypto - 1b-AcpP (C16-AcpP) placed Leu263 within the buried hydrophobic core of KASII-A, distant from the catalytic residues Cys240, H380, and H416 in the active site. FoldX predicted a destabilizing effect of 6.83 kcal/mol for L263F, attributed to van der Waals clashes. Rosetta analysis also indicated a destabilizing energetic change of 11.57 REU. In F₂ populations derived from Pungsannamul × PE1604 and Seonpung × PE1604, palmitic acid composition increased from 12.1% in KASII-A homozygotes to 18.1% in kasII-a homozygotes and 11.7% in KASII-A homozygotes to 17.4% in kasII-a homozygotes, respectively. In addition, analysis of fatty acid content showed that palmitic acid content in kasII-a homozygotes progeny paralleled the difference in palmitic acid content between the parental lines. Seonpung contained approximately 33.5 mg/g seed weight palmitic acid content, and kasII-a homozygotes progeny from the Seonpung × PE1604 cross accumulated 51.9 mg/g seed weight, wherease Pungsannamul conta0ined approximately 23.1 mg/g seed weight and the corresponding progeny from the Pungsannamul × PE1604 cross accumulated 38.2 mg/g seed weight. These results suggest that the L263F amino acid substitution may affect KASII-A through altered local packing and protein stability rather than direct perturbation of the catalytic site.