Structural Prioritization of FatB Thioesterase Candidates Potentially Related to Lauric Acid-Rich Seed Oil in Litsea cubeba

Lauric acid is a characteristic component of Litsea cubeba seed oil, but FatB thioesterase candidates with predicted structural compatibility for C12 acyl-substrate accommodation remain insufficiently defined. In this study, seed oil content and fatty acid composition were examined during L. cubeba seed development. The fatty acid profile shifted from a C18:2-rich pattern at the early stage to a C12:0-dominated composition at later stages, providing the biochemical context for FatB candidate prioritization. Three FatB-like candidates were retrieved from a de novo seed transcriptome assembly and named LcFatB1, LcFatB2, and LcFatB3. Phylogenetic analysis, conserved motif comparison, sequence alignment, and homology modeling showed that LcFatB1 and LcFatB2 retained more complete FatB-like sequence and structural features than LcFatB3. S-dodecanoyl-4′-phosphopantetheine was used as a C12 acyl-4′-phosphopantetheine surrogate for molecular docking. Docking analysis indicated that LcFatB1 and LcFatB2 formed more interpretable C12-bound poses than LcFatB3. Subsequent 150 ns molecular dynamics simulations, free energy landscape analysis, residue–ligand interaction profiling, and catalytic tunnel analysis further distinguished the two main candidates. Compared with LcFatB2, LcFatB1 maintained a lower-displacement C12-bound state, a more compact contact environment involving Tyr116, Ser125, and Asn278, and a main tunnel with higher throughput and shorter length in the representative global-minimum conformation. LcFatB2 also retained the C12 surrogate but stabilized it in a distinct rearranged binding environment. These results support LcFatB1 as the strongest structurally prioritized FatB candidate among the three transcriptome-derived proteins, while LcFatB2 remains a plausible FatB-like candidate with a distinct C12-bound state. This prioritization provides computational structural clues for future biochemical testing but should not be interpreted as direct functional confirmation of FatB activity in vivo.