A Sequence-structure Based Method Developed For Mining New DAG Lipases From Protein Sequence Database

Diacylglycerol?DAG?lipases have received considerable attentions due to their physiological functions and potential usage in oil and fat modification.However,compared to wide application of triacylglycerol?TAG?lipases,industrial application of DAG lipases is still rare due to limitations in their thermostability and specific activity.The structural domains of DAG lipases,the lid and flap domains control the catalytic activity of lipase through the opening and closing motion.However,this gating mechanism of diacylglycerol?DAG?lipase is poorly understood due to the lack of 3D structures in open conformations.In this study,the opening and closing states of Mrlip1 DAG lipase are revealed by the homology modeling and molecular dynamic simulations.It was found that the active residues(Ser₁₇₁,His₂₈₁ and Asp₂₂₈)in the catalytic pocket of Mrlip1 DAG lipase is covered by the lid domain in the closed conformation and exposed to the solvent in the open conformation.The role of residues Phe₂₇₈ and Gln₂₈₂82 in the flap domain,as well as that of Thr₁₀₁01 and Thr₁₀₇ in the lid domains are also identified in gating mechanism.The site-directed mutagenesis has been carried out to illustrate the putative alterations of enzyme specificity.Our results suggest that the substrate specificity is achieved by these two key residues Phe₂₇₈ and Gln₂₈₂,and the irreversible conversion from DAG to TAG?Triacylglycerol?lipase are enabled by the two-point mutations.Moreover,Comparing with TAG lipases,very limited DAG lipases have been reported till date.The four DAG lipases have been discovered so far are all fungal origin,including Penicillium camembertii?PCL?,Aspergillus oryzae?AOL?,both SMG1 and MGMDL2 from Malassezia globose.All of these DAG lipases are mesophilic lipase with rather narrow optimal temperature.Most currently available DAG lipases have preference on sn-1 position and varied preference of chain length.DAG lipases with better specific activity and thermostability are required for industrial application.In this study,we identified structural consensus motifs of DAG lipases that contribute to their DAG specificity on a structural comparison of DAG and TAG lipases.To find potential DAG lipases,sequence motifs and predicted secondary structures were used to screen millions of protein sequences and predict new DAG lipases.In total,83 new putative DAG lipases were identified.The predicted DAG lipases were validated by expression of randomly chosen putative DAG lipases followed by functional assay for DAGs and TAGs substrates specific activity.This newly developed method is efficient and cost effective for discovery of new enzymes.Newly discovered DAG lipases require further characterization to meet potential application needs to full fill the industrial requirements.