| Lipolytic enzymes, including lipases and esterases, are widely present in natural environment. The high yield and easily genetic manipulation have made microbial lipolytic enzymes widely used in many industries such as food production, fine chemistry and pharmaceuticals. With the development of bioinformatics and molecular biology, more and more novel lipolytic enzymes have been discovered and studied. Marine environments with various physiochemical properties lead to the diversity of marine microorganisms in species, gene functions and ecological functions, enabling us to discover a large quantity of novel lipolytic enzymes with unique biochemical characteristics to satisfy industrial application. In this thesis, a novel gene E22 encoding a homoserine transacetylase-like (HTA-like) esterase, was identified from a metagenomic library constructed from deep-sea sediment samples from the Atlantic Ocean. Based on biochemical, structural and mutational analysis, the molecular mechanism for substrate recognition and catalysis of the esterase E22 was studied. We slso suggest that E22 and its homologs represent a new subfamily in the HTA family through analysis on structures, sequences and biochemical properties. In addition, two novel esterase genes, EstD and EstH, were identified from marine bacteria.1. Functional screening for novel marine esterase genesA metagenomic library containing 17,000 fosmid clones was constructed from deep-sea sediment samples from the Atlantic Ocean. By functional screening of this library, a gene E22 encoding a lipolytic enzyme was identified. Sequence analysis showed that E22 shared high similarities with bacterial characterized HTAs, but low similarities with bacterial eaterase. In addition, two novel lipolytic enzyme genes, EstD and EstH, were also identified from the genomes of two marine bacteria. Biochemical analysis indicated that E22, EstD and EstH were all esterases. In summary, three novel lipolytic enzyme genes were identified from marine deep-sea sediment and bacterial genomes.2. Biochemical characterization of esterase E22E22 was heterologously expressed in Escherichia Coli BL21 (DE3) and purified. Its biochemical characterizations were analyzed. The recombinant E22 could efficiently hydrolyze pNP esters with short chains (C2-C6), showing the highest activity towards pNPC4. When pNPC4 was used as the substrate, the specific activity of E22 was 1720 U/mg, and the Km and the kcat/Km was 57.12μM and 3.8×104 s-1 mM-1, respectively. The optimal temperature for E22 activity was 60℃ and the optimal pH was 9. E22 showed limited tolerance to NaCl. Metal ions and EDTA had little effect on the activity of E22, suggesting that E22 may not require metal ions for catalysis. Like other esterases, the activity of E22 was inhibited by phenylmethylsulfonyl fluoride (PMSF).3. Catalytic mechanism of esterase E22To reveal the catalytic mechanism of esterase E22, the crystal structures of E22 and its mutant L374D were resolved at 2.3 A and 1.5 A resolutions, respectively. The structure of L374D was also modeled with p-nitrophenyl butyrate. E22 forms dimers both in crystals and in solution. Monomeric E22 contains two domains, an α/β hydrolase domain and a helical bundle domain (Lid domain). The catalytic triad formed by Ser175, Asp340 and His373 are all located in the α/β hydrolase domain. Based on structural and mutational analyses, the detailed catalytic mechanism of E22 was proposed. The hydrolysis of pNPC4 can be divided into two steps.In the first step, the substrate enters the tunnel and nitro oxygens of its benzoic ring are fixed by Arg294 and Ser72. Simultaneously, an electron from Asp340 is transferred to Ser175 through His373. The activated Ser175 attacks the carbonyl carbon of pNPC4 to form tetrahydral acyl-enzyme intermediate, which is stabilized by the oxyanion hole composed of the backbone amide nitrogens of Phe71 and Met176. Then His373 donates one proton to the intermediate, resulting in the release of p-nitrophenol. Butyrylated Ser175 is formed during the first step. In the second step, a tetrahedral transition state is formed again by the same oxyanion hole. Then His373 obtains a proton from the coming water molecule, and the resulting OH- ion attack the carbonyl carbon of the intermediate to release butyrate, making the enzyme back to the active state.4. Structural differences for substrate recognition between E22 and the other two HTA subfamiliesAlthough E22 and HTAs are similar in sequences and topological structures, E22 could efficiently hydrolyze pNPC6 but showed no transacetylase activity. To reveal the variations between E22 and other two HTA subfamilies, comparative structural analysis on them was carried out. The results indicated that E22 and HTAs have the same catalytic triad, but differ in substrate binding tunnels. Esterase E22 forms a more extensively positively charged tunnel, which does not fit for the binding of the hydrophobic patches of large acetyl-CoA molecules. Four residues involved in the binding of acetyl-CoA conserved in HTAs have been changed to other residues in E22. In addition, two conserved hydrophilic residues in HTAs involved in the binding of homoserine are also replaced by hydrophobic ones in E22.The deeper and hydrophobic tunnel behind catalytic Ser175 in E22 can accommodate esters with acyl length of up to six carbon atoms. The variations in structures make E22 differ from other two subfamilies in HTA. Phylogenetic analysis showed that E22 and its homologs form a distinct group, which are more closely related to HTA subfamily than acetyl esterase subfamily. Substrate specificity analysis showed that three E22 homologs in this group also functioned as esterases and had no detectable transacetylase activity, just like E22, further confirming that E22 and its homologs represent a new subfamily in the HTA family, separate from the bona fide acetyltransferase subfamily and the acetyl esterase subfamily, which is named as the E22 subfamily. |
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