Study On The Mechanism Of Substrate Specificity Of Thermophilic Esterase

Lipase is an important member ofα/βhydrolase family, which can catalyze the hydrolysis of ester bond,and thus participate in lipid metabolism in organism. Structural analysis indicates that lipases possess typicalα/βhydrolase domain as well as conservative catalytic triad. Lipase family members have evolved to three classes to adapt external environment changes as well as the divergence of internal metabolic pathway. Among them, esterase and lipase are the most appealing targets. The main difference between esterase and lipase lies on the substrate specificity: esterases preferentially hydrolyze water-soluble simple esters and usually only triglycerides bearing fatty acids shorter than C6, whereas lipases prefer water-insoluble substrates, typically triglycerides of long-chain fatty acids. Owing to the ability to hydrolyze triglycerides, lipases exhibit important potential to be applied in biodiesel and chemical engineering. However, up to now, there are rare reports on the lipases of good stability and ability to catalytic triglycerides at high temperature. Therefore, to investigate the catalysis mechanism and construct thermophilic lipase of high stability by divergent evolution is the hot-spot in enzyme engineering. Thermophilic esterase AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus, a member of hormone-sensitive lipase family, is a typical thermophilic esterase, and can only catalyze the hydrolysis of short chain pNP ester. In order to search the substrate specificity regulation site of HSL family, we assume AFEST as a progenitor of HSL family to study the substrate specificity and catalytic mechanism through semi-rational design. Based on keeping protein stability, constructing thermophilic lipases and realizing enzyme functional divergent evolution will be beneficial to solve the problem of rare thermophilic lipases in nature evolutionary process and gain deeper insight into molecular evolution mechanism, and thus promote the industrial applications of enzymes.Structures comparison of AFEST and various lipases was performed through bioinformatics methods, and the major factors that limited the lipase activity of AFEST were analyzed. It was inferred that the 33th and 44th amino acids inα2-helix of cap structure were essential for reducing the interactions between the helix and adjacent domain, and thus enhancing the hydrophobicity and driving the movement of cap structure. Therefore, the 33th and 44th amino acids were the key residues for the conversion of substrate specificity. In this thesis, the mutants R33A and N44A were conducted to investigate the importance of amino acids. pNP esters of various chain length and triglycerides are selected to inspect the substrate specificity of R33A and N44A. The results indicated that compared with wild type, the activity of R33A and N44A towards triglycerides has been dramatically improved. In addition, N44A tended to catalyze long chain substrate than R33A. We presumed that N44 was much more important for altering the substrate specificity of AFEST, which meant that N44 was potential to be reconstructed. In consequent, N44 was selected as a hotspot for saturation mutation. Results from saturation mutation library showed that AFEST presented remarkable advanced activity to triglycerides when N44 was replaced by amino acids with small side chain (A, G), with -OH side chain (S, T) and large hydrophobic side chain (P, F). The lipase activity of mutants were obtained based on remaining the esterase activity, which illuminated that the amino acid 44 did not have great influence on esterase activity but had decisive effect for obtaining lipase activity. The major difference between esterase and lipase was whether triglycerides could enter into the catalytic centre through the lid switch and then be hydrolyzed. The mutations have not changed the active center and the substrate binding pocket, and therefore the mutants and wild type had the similar activity towards short chain ester.Compared with wild type, the optimal temperature of the mutants has been decreased slightly and the activation energy was reduced. The thermodynamic stability of enzyme could be represented by melting temperature Tm. The thermal unfolding of each protein is determined by fitting the ellipticity at 222 nm versus temperature, and the Tm values were calculated through two-state model. Tm decreased from 85℃for wild type by 21.5℃for N44A, 14℃for N44S and 30℃for N44F. These results implied that compared with wild type, the thermodynamic stability of mutants was reduced. The flexibility analysis of wild type and mutant after molecular dynamic simulation revealed that when N44 amino acid was replaced by hydrophobic amino acid, the whole structural flexibility was reinforced due to the change of enzyme intramolecular microenvironment. Whereas N44 was replaced by amino acid bearing -OH side chain, the enzyme intramolecular microenvironment did not change much, and therefore no important influence was brought to the whole structural flexibility. These results were consistent with those obtained form circular dichromatism.Furthermore, the effect of N44 on substrate specificity was investigated by molecular dynamics simulation and substrate docking. After molecular dynamics simulation process of 1 ns, it was obviously shown that the mutant in amino acid 44 caused the relative conformation alteration between oxygen hole and catalytic triad. Ala, Ser and Phe could destroy the hydrogen bond disfavoring for oxygen anion hole which contributed to stabilizing the transition complex, then the activation energy was reduced, and thus the reaction velocity was improved. Docking results also showed that the mutants possessed lower reaction activation energy towards triglycerides to promote the reaction velocity than wild type. In addition, N44F had the lowest reaction activation energy, so the catalytic velocity was the highest. The above results coincided with those obtained in the activity measurement.In this thesis, N44 was identified as the key point to acquire lipase activity. This finding could offer evidences for diverge evolution mechanism of HSL lipase family, and then provide a new route for exploring new enzymes.