Two novel deep-sea sediment metagenome-derived esterases: residue 199 is the determinant of substrate specificity and preference

Abstract

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.

Keywords: Deep-sea; Esterase; Family IV; Homology modeling; Metagenomics library; Substrate-binding pocket.

Fig. 1

Neighbor-joining phylogenetic tree based on the amino acid sequences of DMWf18-543, DMWf18-558 and related lipolytic enzymes. Sequence alignment was performed using ClustalX, and the tree was constructed using MEGA software. Bootstrap values are based on 1000 replicates, and only values > 50% are shown. The scale bar indicates the number of amino acid substitutions per site

Fig. 2

Amino acid sequence alignment of DMWf18-543- and DMWf18-558-related lipolytic enzymes. The accession numbers of the enzymes in the GenBank database are given for DMWf18-543 and DMWf18-558 (from this study), Est6 (AFB82690), Est4 (AFB82689), EstMY (ADM67447), and ArmEst1 (AGF91877). Sequence alignment was performed using the ClustalX and ESPript programs. Identical and similar residues among groups are indicated in white text on a red background and in red text on a white background, respectively. Solid circles indicate the locations of the residues involved in the oxyanion hole (glycine (G)). The triangles indicate the locations of the catalytic active site residues (serine (S), aspartate (D), and histidine (H)). The square indicates the location of residue Leu199 of DMWf18-543 and residue Ala199 of DMWf18-558. The conserved HGGG and GXSXG motifs, in which the oxyanion hole and catalytic triad are located, are outlined with boxes

Fig. 3

Characterization of DMWf18-543 and DMWf18-558. a Substrate specificity was determined using the p-NP esters, including p-NP acetate (C2), p-NP butyrate (C4), p-NP caprylate (C8), p-NP decanoate (C10), p-NP laurate (C12), p-NP myristate (C14), and p-NP palmitate (C16). All of the tests were performed at 35 °C and pH 7.5. b Effects of pH on the activity were determined in different buffers: 100 mM citrate buffer (pH 3.0–6.0), 100 mM phosphate buffer (pH 6.0–7.5), 100 mM tricine buffer (pH 7.5–9.0), and 50 mM CHES buffer (pH 9.0–10.0). All of the tests were performed at 35 °C using p-NP butyrate as the substrate. c Effects of temperature on the activity were determined at various temperatures at pH 7.5 using p-NP butyrate as the substrate. The highest activity was taken as 100%. Data are presented as the mean ± SD (n = 3)

Fig. 4

Cartoon representation of 3D structural models of DMWf18-543 (a) and DMWf18-558 (b). The catalytic domains are shown in green/blue and yellow, and the cap domains are shown in magenta. The catalytic triad residues are indicated as stick models

Fig. 5

Substrate-binding pockets of DMWf18-543 and DMWf18-558. Surface view of the structures showing the differences in the substrate-binding pockets of DMWf18-543 (a) and DMWf18-558 (b). The substrate-binding pockets are indicated with red dashed lines. Residues of the catalytic triad are shown as stick models in yellow. c Superposition of the surfaces of DMWf18-543 (green) and DMWf18-558 (gray). Leu199 of DMWf18-543 and Ala199 of DMWf18-558 are shown as stick models

Fig. 6

Substrate specificity of DMWf18-558-A199L. The esterase activity of the mutant was determined using p-NP esters. The highest activity was taken as 100%. Data are presented as the mean ± SD (n = 3)