Structural characteristics and catalysis properties of a dual-functional enzyme Lip10 from Mucor circinelloides

Abstract

Mucor circinelloides lipase Lip10 is a dual-functional enzyme with lipolytic activity and phospholipid:diacylglycerol acyltransferase (PDAT) activity in aqueous phase. However, the catalytic properties of Lip10 and its structural characteristics remain unknown. The present study systematically investigated its PDAT activity and lipolytic activity using six phospholipid substrates, 18:0 triacylglycerol and 16:0 1,2-diacylglycerol, predict the 3D structure of Lip10 using AlphaFold2, and evaluated residues’ flexibility through molecular dynamics analysis. Lip10 exhibited a preference for phosphorylethanolamine (PE) with a long-chain fatty acyl group and showed the lipolytic activity to 18:0 triacylglycerol and 16:0 1.2-diacylglycerol. 20 mM Mg2+ and Fe2+ can significantly increase the PDAT activity of Lip10 by 50–80%. Molecular docking analysis suggest that the lower binding energy for 18:0 PE with Lip10 contributes to the higher structural stability of the complex of 18:0 PE and Lip10. Mutations of Phe565Asp and Glu318Met increase PDAT activity of Lip10 by 3.6-fold and 2.7-fold, while the TAG content produced by mutants Lip10Phe103Pro, Lip10Ile487Lys and Lip10Phe570Asp decrease by 33%− 43%. The present study demonstrated structural properties of the dual-functional lipase Lip10 and provide a theoretical basis to study its roles in the lipid metabolism of M. circinelloides in the future.