## 3 Results  
### Sequence Analysis and Expression of EstDR4  
The _estDR4_ gene (939 bp) that encodes the 312-amino-acid protein EstDR4 from \(D\). _radiodurans_ was inserted into pET-28a for overexpression in _E. coli_ BL21(DE3). BLASTP analysis revealed that EstDR4 had greater than 50% amino acid sequence similarity with some uncharacterized hydrolases, including an \(\alpha/\beta\) hydrolase from _Deinococcus wuuluquiensis_ (81.35%), a lipase from _Deinococcus swuensis_ (67.74%), and an acetyl hydrolase from _Alphaproteobacteria bacterium_ 65-37 (50.00%). EstDR4 showed significant amino acid sequence similarity with esterases from family IV, such as Est8 from a metagenomic library (PDB ID 4 YPV, 44.30%), EstDL136 from a metagenome (PDB ID 6 AAE, 42.24%), and LipP from _Pseudomonas_ sp. B11-1 (AAC38151.1, 44.59%). A phylogenetic tree including the EstDR4 protein and representative members of eight lipolytic bacterial enzyme families was constructed to obtain further insight into the evolutionary relationships of EstDR4 (Figure 1a). Phylogenetic analysis revealed that EstDR4 and its homologs belonged to family IV, of which most of esterases display a striking amino acid sequence similarity to hormone-sensitive lipase (HSL), thus, lipolytic enzyme family IV is also called the HSL family. Multiple sequence alignments of EstDR4 with LipP (AAC38151.1) and Est8 (PDB 4 YPV) revealed that the conserved motifs HGGG and GXSXG, which exist in lipolytic enzyme family IV, were also present in EstDR4 (Figure 1b).  
Recombinant EstDR4 with N-terminal His-tag was successfully expressed and purified (Figure 2b). The tertiary structure of EstDR4 was modeled in accordance with the structure of Est8 (PDB 4 YPV, 44.3%) using the Phyre\({}^{2}\) server. The model comprised an \(\alpha/\beta\) hydrolase fold and a cap domain. The predicted catalytic amino acid triad residues, Ser156, Asp253, and His283, were in spatial proximity to each other (Figure 2a). Three mutant strains were constructed to experimentally confirm the putative catalytic residues; as expected, all three mutants, S156A, D253A, and H283A, showed negligible activity compared to the wild type (Figure 2c).  
Figure 1: Phylogenetic tree of EstDR4 and homologous sequence alignment. (**a**) Phylogenetic evolutionary tree of EstDR4 constructed using 21 amino acid sequences of lipolytic enzymes from different families and EstDR4. The protein ID at NCBI or the protein databank (PDB) are shown before the name of the strain that was the enzyme source. (**b**) Homologous sequence alignment constructed using the amino acid sequences of EstDR4, _Pseudomonas_ sp. lipase (AAC38151.1), and _Pariubaculum_ sp. esterase (PDB ID 4 YPV), which all belong to family IV. The catalytic triads are marked by black arrows.  
Figure 2: Tree dimensional structure, purification and enzyme activity of the mutants of EstDR4. (**a**) The predicted three-dimensional structural model of EstDR4. The tertiary structure of EstDR4 (blue ribbon) was modeled on that of Est8 (PDB 4 YPV, gray ribbon). Potential catalytic triad residues (Ser156, Asp253 and His283) are marked on the figure. (**b**) SDS-PAGE electrophoresis of EstDR4. Lane M: marker, lane 1: crude, lane 2: purified EstDR4. The EstDR4 protein band at \(\sim\)35 kDa, was in accordance with the prediction that EstDR4 (33 kDa) added with the HisTag. (**c**) WT is the wild type EstDR4, the mutants are S156A, D253A and H283A. The different letters between each condition indicate significant differences (\(p<0.05\)).