Esterases are a class of enzymes that split esters into an acid and an alcohol in a chemical reaction with water, having high potential in pharmaceutical, food and biofuel industrial applications. To advance the understanding of esterases, we have identified and characterized E53, an alkalophilic esterase from a marine bacterium Erythrobacter longus. The crystal structures of wild type E53 and three variants were solved successfully using the X-ray diffraction method. Phylogenetic analysis classified E53 as a member of the family IV esterase. The enzyme showed highest activity against p-nitrophenyl butyrate substrate at pH 8.5 9.5 and 40 C. Based on the structural feature, the catalytic pocket was defined as R1 (catalytic center), R2 (pocket entrance), and R3 (end area of pocket) regions. Nine variants were generated spanning R1-R3 and thorough functional studies were performed. Detailed structural analysis and the results obtained from the mutagenesis study revealed that mutations in the R1 region could regulate the catalytic reaction in both positive and negative directions; expanding the bottleneck in R2 region has improved the enzymatic activity; and R3 region was associated with the determination of the pH pattern of E53. N166A in R3 region showed reduced activity only under alkaline conditions, and structural analysis indicated the role of N166 in stabilizing the loop by forming a hydrogen bond with L193 and G233. In summary, the systematic studies on E53 performed in this work provide structural and functional insights into alkaliphilic esterases and further our knowledge of these enzymes.
Title: Two novel deep-sea sediment metagenome-derived esterases: residue 199 is the determinant of substrate specificity and preference Huo YY, Cheng H, Rong Z, Cui HL, Xu XW Ref: Microbial Cell Factories, 17:16, 2018 : PubMed
Background
The deep-sea environment harbors a vast pool of novel enzymes. Owing to the limitations of cultivation, cultivation-independent has become an effective method for mining novel enzymes from the environment. Based on a deep-sea sediment metagenomics library, lipolytic-positive clones were obtained by activity-based screening methods.
Results
Two novel esterases, DMWf18-543 and DMWf18-558, were obtained from a deep-sea metagenomic library through activity-based screening and high-throughput sequencing methods. These esterases shared 80.7% amino acid identity with each other and were determined to be new members of bacterial lipolytic enzyme family IV. The two enzymes showed the highest activities toward p-nitrophenyl (p-NP) butyrate at pH 7.0 and 35-40 C and were found to be resistant to some metal ions (Ba2+, Mg2+, and Sr2+) and detergents (Triton X-100, Tween 20, and Tween 80). DMWf18-543 and DMWf18-558 exhibited distinct substrate specificities and preferences. DMWf18-543 showed a catalytic range for substrates of C2-C8, whereas DMWf18-558 presented a wider range of C2-C14. Additionally, DMWf18-543 preferred p-NP butyrate, whereas DMWf18-558 preferred both p-NP butyrate and p-NP hexanoate. To investigate the mechanism underlying the phenotypic differences between the esterases, their three-dimensional structures were compared by using homology modeling. The results suggested that residue Leu199 of DMWf18-543 shortens and blocks the substrate-binding pocket. This hypothesis was confirmed by the finding that the DMWf18-558-A199L mutant showed a similar substrate specificity profile to that of DMWf18-543.
Conclusions
This study characterized two novel homologous esterases obtained from a deep-sea sediment metagenomic library. The structural modeling and mutagenesis analysis provided insight into the determinants of their substrate specificity and preference. The characterization and mechanistic analyses of these two novel enzymes should provide a basis for further exploration of their potential biotechnological applications.
Lysophospholipase_carboxylesterase (LPCE) has highly conserved homologs in many diverse species ranging from bacteria to humans, as well as substantial biological significance and potential therapeutic implications. However, its biological function and catalytic mechanism remain minimally investigated because of the lack of structural information. Here, we report the crystal structure of a bacterial esterase PE8 belonging to the LPCE family. The crystal structure of PE8 was solved with a high resolution of 1.66 A. Compared with other homologs in the family, significant differences were observed in the amino acid sequence, three-dimensional structure, and substrate-binding pattern. Residue Arg79 undergoes configuration switching when binding to the substrate and forms a unique wall, leading to a relatively closed cavity in the substrate-binding pocket compared with the relatively more open and longer clefts in other homologs. Moreover, the mutant Met122Ala showed much stronger substrate affinity and higher catalytic efficiency because less steric repulsion acted on the substrates. Taken together, these results showed that, in PE8, Arg79 and Met122 play important roles in substrate binding and the binding pocket shaping, respectively. Our study provides new insight into the catalytic mechanism of LPCE, which may facilitate the development of structure-based therapeutics and other biocatalytic applications.
