A mutant T1 lipase homology modeling, and its molecular docking and molecular dynamics simulation with fatty acids
Introduction
Fatty acids (FAs) are major components of natural fats and oils, and they have different effects on human health. For example, caprylic acid (C8:0) which is released after fats and oils digestion can be rapidly assimilated for energy (Wallace, 2019) making it potentially valuable for medical and nutraceutical applications, and linolenic acid (C18:3) can reduce the concentration of low density lipoprotein cholesterol when it replaces the major dietary saturated FAs, thus reducing cardiovascular risk (Calder, 2015). In general, most natural fats and oils are composed of different chain-length FAs, thereby resulting in low content of certain FAs. Therefore, it has become increasingly valuable to enrich desirable FAs in the form of acyglycerols.
Lipase-catalyzed selective esterification, hydrolysis and interesterification are promising methods to concentrate desirable FAs in the form of acyglycerols due to its eco-friendly process, mild reaction conditions and selectivity. T1 lipase from Geobacillus zalihae strain is an excellent thermostable lipase with optimal activity at 65–75 °C (Leow et al., 2007), which is a potential industrial biocatalyst as it would allow the enzymatic reactions to be performed at high temperature and thereby lower substrate viscosity, improve substrate solubility, and increase diffusion coefficient and reaction rate. In addition, T1 lipase shows broad substrate specificity but with different preference for fats and oils (Leow et al., 2007), and exhibits 1,3-regio-selectivity (Qin et al., 2014b; Xu et al., 2013), which is potential candidate of interest for the modification of fats and oils. Up to now, many strategies have been developed to optimize the lipase production and meet commercial demand, including enhanced enzyme production in a novel system (Abu et al., 2020), enhanced purification through molecular modification (Hussian et al., 2018), structure-function relationship (including activity and stability against high temperature, pH and organic solvents) studies utilizing site-directed mutagenesis (Ishak et al., 2019; Ruslan et al., 2012; Tang et al., 2017), and catalytic mechanism (Rahman et al., 2012; Wang et al., 2010). In addition, wild-type and mutant T1 lipase could catalyze the production of diacylglycerol (Xu et al., 2013), FA sugar ester (Abdulmalek et al., 2020), structured lipids (Qin et al., 2014a) and menthyl butyrate (Wahab et al., 2014). However, there are limited reports on interactions between the lipase and the aliphatic substrates. Understanding of the lipase-substrate interaction may provide further insight into the conformational changes of T1 lipase, which is useful for rationally designing binding sites of T1 lipase with substrates of interest and applications in enzymatic production of acyglycerol enriched with some FAs of interest.
Molecular docking and molecular dynamics (MD) simulation methods have been used to predict interactions of enzyme with ligand (Afzal et al., 2014; Anuar et al., 2020; Hou et al., 2020). The interactions between T1 lipase and selected chemical ligands with aromatic rings, amine and hydroxyl groups were explored by using molecular docking, providing structural information for the discovery of novel semisynthetic enzymes (Rahman and Latif, 2019). To date, there are few reports regarding interaction mechanism of T1 lipase with FAs at the molecular level using molecular docking and MD simulation studies. Our previous studies showed that T1 lipase fused to cellulose-binding domain could catalyze the hydrolysis of palm stearin to produce diacylglyrerols (Xu et al., 2013), the acidolysis of palmitin-enriched triacylglycerols to synthesize medium-long-medium structured lipids (Qin et al., 2014a), and the esterification of glycerol with FAs to synthesize acylglycerols (Qin et al., 2014b). Furthermore, the effect of hydrophobic residues in the lid of wild-type T1 lipase and mutant T1 lipase (Mut-T1 lipase) on 1,2-dilaurin in hydrolysis process was studied, and molecular docking results indicated that the residues of Leu183 and Val187 were involved in binding to 1,2-dilaurin (Xin, 2017). These works demonstrated that the Mut-T1 lipase would be a potential biocatalyst in the modification of fats and oils. Study on interactions between T1 lipase (wild type and/or mutant form) and substrates at the molecular level will help understand the development and applications of Mut-T1 lipase. In this work, a two-step framework by combining molecular docking and MD simulation was performed to investigate the detailed interactions between the Mut-T1 lipase and aliphatic substrates. Firstly, structure optimization and molecular properties of selected FAs were calculated. Secondly, the 3D structure of lipase was predicted by homology modeling. Subsequently, the possible interactions of the modeled structure of Mut-T1 lipase and optimized structure of FAs were analyzed by molecular docking and MD simulation. This computational workflow could provide important insight into structural characteristics of the lipase bound to various FAs and help to enhance the discovery of novel semisynthetic enzymes as new biocatalyst.
Section snippets
Structure optimization and molecular properties calculation of fatty acids
Firstly, C8:0, myristic acid (C14:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and C18:3 were selected as substrates. Molecular structures of the six types of FAs were constructed using GaussView 5.0.9. Secondly, the structure optimization and vibrational frequency calculation for the six FAs under vacuum were completed with Gaussian 09 software (Frisch et al., 2009) at the B3LYP-D3(BJ)/6-311G(D,P) level of density functional theory. Imaginary frequency was not found for
Structural and electronic properties of FAs
Geometry optimization is the first important step (Schlegel, 2011) in molecular docking and MD simulation related to the structure and reactivity of FA molecules. After optimization, the frontier orbitals (HOMO and LUMO) and HOMO-LUMO energy gaps for optimized geometry of FAs were computed through single-point calculations. Fig. 1 shows the optimized geometries of FAs and their electronic properties. All the saturated FAs (i.e., C8:0, C14:0 and C18:0) were linear molecules with sp3
Conclusion
The study built the 3D structure of Mut-T1 lipase by homology modeling, and assessed the interactions between Mut-T1 lipase and selected FAs by employing molecular docking and MD simulation. Molecular docking showed that binding affinity of FAs towards Mut-T1 lipase was high as the number of carbon atoms and double bonds of acyl chain increased. However, the conformation of Mut-T1 lipase-C18:1 complex being comparably unstable during MD simulation, in terms of high root-mean square fluctuation.
CRediT authorship contribution statement
Xiaoli Qin: Methodology, Writing - original draft, Funding acquisition, Formal analysis. Jinfeng Zhong: Methodology, Software, Data curation, Writing - review & editing, Formal analysis. Yonghua Wang: Conceptualization, Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
This research was funded by the Key Program of Natural Science Foundation of China (No. 31930084), Natural Science Foundation of Chongqing (No. cstc2019jcyj-msxmX0113), National Key R&D Program of China (No. 2018YFC0311104), and National Outstanding Youth Science Foundation of China (No. 31725022).
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