## 4 Discussion  
Extremophiles that thrive in extreme adverse environmental conditions are regarded as valuable sources of novel enzymes required in industrial applications [9]. As a polyextremophile bacterium, _D. radiodurans_ is well known for its radiation resistance [10], and can withstand oxidative stress, pressure, and low temperatures [11; 12; 13]. With interest in isolating lipolytic enzymes from extremophiles, one of the putative lipolytic enzyme genes (_estDR4_) in _D. radiodurans_ was cloned, expressed and characterized. Sequence analysis indicated that EstDR4 belongs to family IV of bacterial lipolytic enzymes, also called the HSL family. The predicted catalytic amino acid triad residues, Ser156, Asp253 and His283, were confirmed by site-directed mutagenesis. Two of the catalytic residues, Ser156 and Asp253, were located in the typical motifs of HSL family esterases, pentapeptide GDSAG and C-terminal conserved motif DPLR, respectively. The catalytic residue His283 was located in HGY, which differs from the common type HGF in HSL family esterases; the functional significance of this variant is currently unknown. Moreover, this study has confirmed that EstDR4 has a weak hydrolysis effect on TA. EstDR4 contained a GGGX type (82 HGGG85) oxyanion hole, which has been proposed to be responsible for the ability of these enzymes to hydrolyze tertiary alcohol esters [14; 15]. This motif is also present in esterases from other sources such as RmEstA from _Rhizomucor miehei_ and \(\gamma\)-PLE, an isomerase of pig liver esterase [16; 17]. The tertiary structure of EstDR4 was modeled according to the structure of Est8 (PDB 4 YPV), an alkaline esterase from a metagenomic library [18]. The tertiary structure of EstDR4 exhibited a cap domain and \(\alpha/\beta\) hydrolase fold. Lipolytic enzymes of HSL family have various substrate preferences, such as LipU from _Mycobacterium tuberculosis_ H37 Rv toward \(p\)NPC4, Est06 from a forest soil metagenome toward \(p\)NPC5, and thermal stability lipase Blip from _Bacillus halodurans_ prioritizes medium to long chain fatty acyl esters [19; 20; 21]. Est8 shares 44.3% amino acid sequence similarity with EstDR4, although they have quite different substrate preferences, with EstDR4 favoring \(p\)NPC8, and Est8 favoring \(p\)NPC2. Similar to Est8, the tertiary structure of EstDR4 revealed spatially adjacent catalytic residues, which might not accommodate long-chain esters. Thus, the difference in substrate preference may be due to differences in substrate channel and cap domain. Two aromatic residues in Est8, Met213 and Phe217, were observed to block the substrate channel, and mutations Met213 Gly and Phe217 Val led to increasing relative activity against C4 and C5 [18]. The corresponding residues in EstDR4 are Met212 and Gly216, and a smaller side chain in site 216 might be a reason for mid-chain substrate preference. Moreover, the cap domain of EstDR4 contains two shorter \(\alpha\)-helices than in Est8 (Supplementary Figure S1), which may allow longer substrates to enter the active center. EstDR4 catalyzes the hydrolysis of a variety of substrates containing phenyl ester, tertiary alcohol and triglyceride, and the regioselectivity and enantioselectivity were identified by asymmetrical hydrolysis of \(\alpha\)-NA, \(\beta\)-NA, (R)-MHM and (S)-MHM. These identify EstDR4 for potential use in a wide range of industrial applications. EstDR4 showed optimum activity at 30 \({}^{\circ}\)C, which concurs with the reported maximum activity of other cold-adapted esterases, such as EstT1-39 from the psychotolerant _Pseudomonas_ sp. T1-39, EstLiu from _Zunongwangia profunda_, PMGL3 from the permafrost microbial community and rEstSL3 from _Alkalibacterium_ sp. SL3 [22; 23; 24; 25]. While EstDR4 had higher relative activity at an environment below 10 \({}^{\circ}\)C compared with cold-adapted esterase PMGL3 [24] and PsyEst from _Psychrobacter_ sp. Ant300, another cold-active esterase [26].