Structural insights into thraustochytrid-specific lipases using alphafold to identify the role of GXSXG motif

Triacylglycerol lipases (E.C. 3.1.1.3) are serine hydrolases, universally present in animals, plants and microbes and are an integral part of lipid metabolism. They are industrially relevant enzymes that cleave ester bonds of triacylglycerides to release free fatty acids and glycerol. Thraustochytrid Aurantiochytrium limacinum SR21 has previously been reported to utilize 120 g L^- 1 of oil substrate. Previously, thraustochytrid specific lipases was reported that allowed the microbe to thrive on oil substrate, however the structural characteristics of these enzymes remain undetermined.

In this study, we identified nearly 30 genes that encode TAG lipases with Lipase_3 domain, allowing the marine microbe to thrive on oil substrate. The lipases were predicted to localize at several subcellular compartments such as extracellular (31293), membrane-bound and cytosolic. Phylogenomic analysis revealed that lipases from thraustochytrids form distinct clades, diverging significantly from the well-characterized lipases from yeast Yarrowia lipolytica. Motif enrichment analysis confirmed the presence of the conserved 'GXSXG' motif in all lipases, where serine serves as the catalytic residue. Notably, histidine (H) or tyrosine (Y) was found at the second position of the motif in A. limacinum SR21 lipases 34357 (cytosolic) and 31293 (extracellular) respectively, suggesting functional differences. Docking analysis with tripalmitoylglycerol (4RF) revealed lower binding energy (ΔG = -5.7 kcal/mol) for cytoplasmic lipase 34357, indicating a stronger ligand interaction compared to ΔG = -3.4 kcal/mol for the extracellular lipase 31293. This suggests that substituting histidine for tyrosine in the active site affects lipase catalytic efficiency and substrate specificity.

Our study provides novel insights regarding the structure and ligand binding affinities for thraustochytrid specific lipases which are diversified attributed to the heterogeneity within the catalytic triads. In conclusion, we hypothesize that differential localization and higher binding efficiency of thraustochytrid specific lipases allow the microbe to efficiently utilize oil substrates. These thraustochytrid-specific lipases are potential candidates for commercialization as large-scale production of thraustochytrids can be achieved sustainably by cultivating on sustainable substrates and these enzymes are highly efficient and robust.