Diacylglycerol acyltransferase (DGAT) is a membrane-embedded, acyl-CoA-dependent enzyme that catalyzes the terminal and rate-limiting step of neutral lipid biosynthesis. However, the structural determinants governing its catalytic function remain poorly defined, hindering rational engineering of this enzyme family. Here, we characterized Saccharomyces cerevisiae DGAT (ScDGA1) through integrative structural prediction, molecular dynamics (MD) simulations, and substrate-docking analyses, and provide the first mechanistic evidence that dynamic tunnel opening governs acyl-CoA access to the catalytic center. Alanine scanning identified Q282 as a key constriction point within the acyl-chain tunnel, and saturation mutagenesis yielded the Q282F variant, which substantially enhanced triacylglycerol accumulation in vivo (1.85-fold, 148 mg/g DCW). Biochemical assays revealed increased lipid droplet biogenesis and a substrate preference shift toward oleic acid. MD analyses further showed that Q282F reduces internal water occupancy, enlarges tunnel geometry, and stabilizes open conformations, thereby lowering the energetic barrier for substrate diffusion. These findings elucidate the molecular basis of DGAT catalysis and establish a structure-guided framework for engineering acyltransferases toward customized lipid biosynthesis.

• PMID: 41655915
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Journal Article

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Li Y, Zhang C, Huang H, Xu X, Liu Y, Du G, Chen J, Li Z, Lv X, Liu L

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