The esterase gene
from the thermophilic bacterium
sp. K91 was cloned and expressed in
. The monomeric enzyme exhibited a theoretical molecular mass of 24.5 kDa and an optimal activity around 50℃ at pH 9.0. A model of Est8 was constructed using a hypothetical YxiM precursor structure (2O14_A) from
as template. The structure showed an α/β-hydrolase fold and indicated the presence of a typical catalytic triad consisting of Ser-11, Asp-182, and His-185, which were investigated by site-directed replacements coupled with kinetic characterization. Asp-182 and His-185 residues were more critical than the Ser-11 residue in the catalytic activity of Est8. A comparison of the amino acid sequence showed that Est8 could be grouped into the GDSL family and further classified as an SGNH hydrolase. Est8 is a new member of the SGNH hydrolase subfamily and may employ a different catalytic mechanism.
Esterases (E.C. 126.96.36.199) are ubiquitous enzymes with important physiological and biotechnological roles in the synthesis or hydrolysis of ester-containing compounds
. Esterases hydrolyze at least partly soluble fatty-acid esters with acyl chain lengths of less than 10 carbon atoms, whereas lipases display maximal activity toward waterinsoluble long-chain triglycerides
. Common esterases possess the highly conserved motif Gly-X-Ser-X-Gly, and the catalytic Ser residue is inside it.
The GDSL-motif-like (pfam PF00657) subfamily is characterized by the presence of a distinct GDSL motif that differs from the classical GxSxG motif. Some of the GDSL family esterases are further classified into a new subfamily, known as SGNH hydrolases owing to the presence of four invariant catalytic residues [Ser, Gly, Asn, and His (SGNH)] in four conserved blocks (I, II, III, and V), which are important for enzyme activity
. The main chains of the conserved catalytic Ser and Gly residues in blocks I and II and the side chain of Asn in block III serve as the proton donors for the oxyanion hole, which is a positively charged pocket that activates the carbonyl and stabilizes the negatively charged oxyanion of the tetrahedral intermediates
. The His residue acts as a base to make the active site Ser more nucleophilic by deprotonating the hydroxyl group, whereas the Asp, also from block V, is speculated to affect the catalytic His residue by increasing its basic character, stabilizing it during the formation of the tetrahedral intermediate, or ensuring its correct orientation
The SGNH superfamily hydrolases have broad substrate specificities because of the flexibility of the active site of the enzymes, such as acyl-CoA esters
, complex polysaccharides
, and other compounds
. However, the natural substrates for most GDSL lipases remain unknown. To date, numerous GDSL lipases have been identified in plants, and the functions of a few lipases have been demonstrated
. GDSL lipases are involved in plant defense, plant tolerance to environmental stresses, and the metabolism of cutin and wax
. Limited reports on the isolation and characterization of GDSL hydrolases, especially the SGNH superfamily hydrolases from bacteria, are available. The functions of SGNH hydrolases from bacteria need to be further explored.
In this work, we report the identification and characterization of
, a new esterase belonging to the SGNH superfamily. Est8 was characterized in terms of catalytic properties, such as optimal temperature and pH, substrate specificity, and mutation effects of the conservative catalytic triad. A plausible 3D structure is also proposed and compared with known structures.
Materials and Methods
- Chemicals and Reagents
Acrylamide, glycerol, and Luria–Bertani (LB) growth medium were obtained from Fisher Scientific. Nickel-NTA agarose was purchased from Qiagen (Valencia, CA, USA). Fast
DNA polymerase and the
-E2 expression kit were provided by TransGen Biotech (Beijing, China). Isopropyl-β-D-1-thiogalactopyranoside (IPTG), phenylmethanesulfonyl fluoride (PMSF), diethylpyrocarbonate (DEPC), esterase substrates, and all other reagents and chemicals were acquired from Sigma (St. Louis, MO, USA) unless otherwise stated.
- Enzyme Source
gene was amplified
PCR from the genomic DNA of the thermophilic bacterium
sp. K91 using primers 5’-GCAAATCATATTTATCTTGC-3’ (N-terminal) and 5’-CCTTTCTTTGATGATCGATTC-3’ (C-terminal). Direct ligations of PCR products to the
-E2 expression vector were performed according to the manufacturer’s instructions. The ligation mix was then transformed into
BL21 (DE3) cells (Novagen). Individual constructs were isolated from transformants, screened for the correct size insert, and completely sequenced to confirm their nucleotide identity. The transformed strains were grown in LB medium containing 100 µg ampicillin/ml at 37℃ until the OD
reached 0.6. Following induction with 0.7 mM IPTG at 20℃ for 20 h, the cells were harvested by centrifugation.
- Protein Purification and Detection, and Kinetic Assay for Esterase Activity
Cells were harvested by centrifugation at 6,000 ×
for 15 min at 4℃, washed with sterile distilled H
O, and resuspended in sterilized ice-cold buffer A (20 mM Tris-HCl and 0.5 M NaCl; pH 7.2). The cells were disrupted by sonication (7 sec, 150 W) on ice for several times and centrifuged at 10,000 ×
for 10 min at 4℃. The supernatant was applied to a Ni
-NTA agarose gel column for purification, with a linear imidazole gradient of 20 to 500 mM in buffer A. Protein concentrations were determined by the Bradford method using bovine serum albumin as the standard. Detection of the Histagged
was carried out by western blot analysis (transfer buffer: 192 mM glycine, 25 mM Tris base, and 20% methanol, pH 8.0). Monoclonal His-tag antibody (IgG2b), peroxidase-conjugated goat anti-mouse IgG (H+L), and a Super Signal West Pico k it (Thermo Scientific Pierce) were used, and procedures were conducted according to the methods reported by Yang
Est8 activity was assayed with
) acetate as the substrate following the method of Zaide
. Absorbance was measured at 405 nm for the appearance of released
-nitrophenol. One unit of esterase activity (U) was defined as the amount of enzyme required to release 1 µmol
-nitrophenol from the substrate per minute.
substrate with a concentration of 0.1 to 1.2 mM was used to determine the K
. The kinetic parameters were determined by nonlinear curve fitting from the Lineweaver–Burk plot, and analyses were performed at least in triplicate
Different lengths of
-NP esters, namely
-NP acetate (
-NP butyrate (
-NP caproate (
-NP caprylate (
-NP caprate (
-NP laurate (
-NP myristate (
-NP palmitate (
), were used to determine substrate specificity.
- Site-Directed Mutagenesis
Site-directed mutagenesis was performed using a fast mutagenesis system kit (TransGen, China) with the following primers (modified codons underlined): 5’-ATCTTGCCGGCGAT
ACTGTTCAAACG-3’ and 5’-
ATCGCCGGCAAGATAAATATGATTTG-3’ for the S11A mutant; 5’-CAGAAGGGATTAAT
TACACGCATTTTAC-3’ and 5’-
ATTAATCCCTTCTGAAATCATAAAAT-3’ for the D182A mutant; and 5’-ATTAATGATTACACG
TTTACAAAAAAAG-3’ and 5’-
CGTGTAATCATTAATCCCTTCTGAAAT-3’ for the H185A mutant. DNA manipulations were performed according to the manufacturer’s instructions. All mutation sites were confirmed by DNA sequencing. For expression, plasmids were transformed into
BL21 (DE3) cells.
- Sequence Analysis
Whole-genome sequencing of K91 is in progress at the Beijing Genomics Institute (Guangzhou, China). To date, a partial genomic sequence has been obtained. The full-length esterase gene,
, was revealed based on the prediction of open reading frames (ORFs) from the partial genomic sequence by the GeneMark.hm online tool (ver. 2.8;
). Protein similarity searches and alignment were performed using the data from Clustal-W
, and only esterases with structural information in the Protein Data Bank were used. Model building was developed by the SWISS-MODEL and Swiss-Pdb Viewer programs (
using the Protein Data Bank Accession No. 2O14_A (hypothetical YxiM precursor from
) as template, which has a sequence identity of 35% to Est8. ESPript output was used to render the analysis of multiple sequence alignments
- Nucleotide Sequence Accession Numbers
The nucleotide sequences of the 16S rRNA and
gene were deposited in the GenBank database under the accession numbers KJ131181 and KJ131182, respectively.
Results and Discussions
- Sequence Analysis of Est8
A 654 bp ORF that encoded for a 217 amino acid protein was found. A comparison of the partial 16S rRNA with that deposited in the GenBank database showed 100% nucleotide identity to the
A3 type strain (Accession No. GU301908.1), indicating that
sp. K91 was a
strain. The deduced protein was compared with three characterized GDSL-like enzymes available in the Protein Data Bank (PDB; NCBI database) by multiple sequence alignment. Est8 had 35%, 28%, and 27% amino acid sequence identities to the hypothetical YxiM precursor from
(PDB code 2O14_A), AaRGAE (rhamnogalacturan_acetylesterase) from
(PDB code 1DEO_A), and thioesterase I/protease I/lysophospholipase L1 from
(PDB code 1IVN_A), respectively.
shows that the amino acid sequence of Est8 possessed the following features: a putative Sercontaining active GDSL-like motif close to the N-terminus (block I) and the presence of four highly conserved blocks shared with SGNH proteins as previously reported by Upton and Buckley
. These results indicate that Est8 represents another rare example of the SGNH hydrolase family.
Multiple amino acid sequence alignments of Bacillus sp. K91 esterase (Est8) with some members of the SGNH family. ESPript outputs were obtained from Est8, hypothetical YxiM precursor (PDB code 2O14_A), AaRGAE (PDB code 1DEO_A), and thioesterase I/protease I/lysophospholipase L1 (PDB code 1IVN_A) sequences from the PDB databank and aligned with Clustal-W. Residues strictly conserved among groups are shown in white font on a red background. Four conserved sequence blocks (blocks I–V) found in all SGNH hydrolases are boxed. Block I has the characteristic GDS sequence motif. Block II has a Gly as the only conserved residue in the members of this family. GXND is the consensus sequence in block III. Finally, block V has the DXXHP conserved sequence. Residues conserved within a group but showing significant differences between groups are shown in red font. The symbols above the blocks of sequences represent the secondary structure. Triangles represent the locations of the active sites.
- Biochemical Characterization of Est8
The crude enzyme extracted from recombinant
BL21 (DE3) cells was purified using Ni
-NTA metalchelating affinity chromatography (
A), and the presence of Est8 after purification was further confirmed by western blot analysis (
was the best substrate for the enzyme Est8 according to the highest maximum initial velocity (k
). The catalytic efficiency toward
was approximately 207-fold higher than toward
. No significant esterase activity was observed for the substrates with a chain length ≥C
. This observation confirms the assumption that the enzyme Est8 is an esterase rather than a lipase.
SDS-PAGE and western blot detection of Est8. (A) Lane M, molecular mass marker (kDa); lane 1, supernatant (crude extract); lane 2, precipitation; lane 3, purified Est8. (B) Western blotting was used to detect Est8 protein in lanes 1, 2, and 3 in subpanel (A), using anti-His antibody.
Kinetic parameters for Est8 activity onp-NP esters with different chain lengths (C2–C16) at pH 9.0 and 50℃.
Reactions were conducted in triplicate in 50 mM Tris-HCl buffer, pH 9.0, at 50℃, using different chain lengths of substrates over a concentration range of 0.1 to 1.2 mM (n = 3, ± SD).
The optimum temperature for Est8 activity was 50℃, where it was stable. Est8 lost most of its activity when the temperature was more than 90℃; however, it retained about 20% activity at 100℃ (
A). Est8 activity at 50℃ was represented as 100%.
B shows that the remaining activities of Est8 were about 85%, 35%, and 36% of the initial activity after incubation at 37℃, 50℃, and 60℃ for 60 min, respectively. The purified enzyme displayed high activity at alkaline pH and retained more than 80% of its maximal activity at pH 8.5 to 9.5 (
C). Est8 showed maximum activity at pH 9.0 and was represented as 100%, which is higher than most of the thermophilic esterases reported. The thermophilic esterases from
showed maximum activity at pH 7.0
at pH 8.0, and
at pH 7.0 to 8.0
. By contrast, the enzyme was not stable at pH 4.0 to 7.0 (
D). The presence of Co
, and Cu
at 1 mM concentration promoted Est8 activity, but the presence of Ni
, and Ag
inhibited Est8 activity (
A). Est8 activity was strongly activated by CTAB and urea (1 mM) and in 1% organic solvents (ethanol, cyclohexane, n-hexane, and isopropanol). Furthermore, Est8 was activated by Tween-80 at 1% or 5% concentration. DTT inhibited Est8 activity at a concentration of 1 mM, and this inhibition was stronger at higher concentrations (10 mM) (
C). These results indicate that Est8 is suitable for application in a water-restricted medium, such as organosynthetic reactions. Therefore, Est8 can be applied in the industrial field, such as in the synthesis of short-chain esters, flavor, and fragrance esters. The exact mechanism of the physiological function of Est8 is not known, as with most reported esterases.
Biochemical characterization of purified Est8. (A) The effect of temperature on Est8 activity was determined using the p-NPC2 assay in the temperature range of 20℃ to 80℃ in 50 mM Tris-HCl buffer (pH 9.0). (B) Thermostability assay. The enzyme was incubated at 37℃, 50℃, and 60℃ for the indicated times. Residual activity was determined by performing the p-NPC2 assay. (C) Effect of pH on Est8 activity. Reactions were performed in phosphate (pH 6.5 to 7.5), Tris-HCl (pH 7.5 to 9.5), and boric acid (pH 9.5 to 12.0) buffers. (D) pH stability assay. After pre-incubation of the enzyme in buffers from pH 2.0 to 12.0, aliquots were removed at specific time points for the measurement of residual activity at 37℃. Reactions were performed in citrate phosphate buffer for pH 2.0 to 5.0, and other buffers were used as described above. Data represent the mean ± SD of three independent experiments, n = 3.
Effects of metal ions, organic solvents, and chemical reagents on Est8 activity. (A) Differentmetal ions (1 mM) were added individually to the reaction assay in 50 mM Tris-HCl buffer (pH 9.0) at 50℃, and the residual activities were determined using the standard assay with p-NPC2. (B) Effects of 1% and 5% concentrations of organic solvents on Est8 activity. (C) Effects of 1 and 10 mM chemical reagents on Est8 activity. Data represent the mean ± SD of three independent experiments, n = 3.
- Site-Specific Replacements
The conserved amino acid residues of the proposed catalytic triad of Est8 were identified within the GDSL motif at sequence positions 11 (Ser), 182 (Asp), and 185 (His), which form part of the conserved sequence motif DXXH using sequence alignment (
, solid triangle). The inhibition effect of chemical modifiers was determined to investigate the catalytic mechanism of the amino acids Ser11 and His185. PMSF, a Ser-modifying-mechanismbased inhibitor that determines the presence of the nucleophilic Ser at the active site, has been successfully applied to hydrolytic GDSL proteins. Treatment of purified Est8 with 1, 10, and 50 mM PMSF led to decreases in ester hydrolysis activity to about 77.40 ± 0.21%, 57.03 ± 2.97%, and 11.80 ± 0.21%, respectively (
). Thus, a serine residue of the GDSL protein may be part of the catalytic center in Est8. Using the same method, Est8 activity decreased to about 62.47 ± 4.38%, 42.26 ± 2.01%, and 29.27 ± 1.95% after treatment with 1, 10, and 50 mM DEPC, respectively (
). These results suggest that serine and histidine are located at (or near) the esterase catalytic site and may be involved in the activity mechanism.
Inhibition of Est8 enzyme activity by the serine (Ser) inhibitor phenylmethylsulfonyl fluoride (PMSF) and histidine (His) modifier diethylpyrocarbonate (DEPC).
aValues represent the mean ± SD (n = 3) relative to the untreated control samples.
Site-directed mutagenesis was used to replace these three amino acid residues by alanine to further confirm that Ser11, Asp182, and His185 may play important roles in the catalytic activity. Overproduction of the engineered proteins in
BL21 (DE3) cells and their purification were monitored by SDS-PAGE and western blot analysis using an anti-His
antibody, respectively (Figs. S1A and S1B). The activities of the enzyme variants S11A, D182A, and H185A were 21.35 ± 1.87%, 19.96 ± 0.76%, and 13.6 ± 2.53%, respectively, compared with the WT enzyme (Table S1). Replacement of the catalytic Asp182 or His185 residues led to the greatest loss of specific activity. We constructed S11A and D182A, and D182A and His185A double mutants. Replacements of S11A and D182A resulted in an enzyme with 11.57 ± 0.75% residual activity compared with the WT enzyme, and D182A and His185A double mutants retained only 3.89 ± 0.76% residual activity (Table S1). These results suggest that D182 and H185 were the critical catalytic residues of Est8.
Under the same conditions as previously described, the Michaelis–Menten parameters of each Est8 variant were determined with
as substrate. The k
of a wild-type enzyme was approximately 33-fold, 20-fold, and 10-fold higher than that of His185A, D182A, and Ser11A, respectively (
). The unexpected mutation indicates that the potential catalytic triad deduced from known GDSL lipases may not be the critical amino acids involved in the lipolytic reaction of Est8. Therefore, Est8 may employ an unknown catalytic motif that is involved in the specific lipolytic activity.
Kinetic parameters of Est8 enzyme and mutant variants.
Activity was assayed in triplicate, using p-nitrophenyl acetate as the substrate with concentration ranges from 0.1 to 1.2 mM, in 50 mM Tris-HCl buffer, pH 9.0, at 50℃ (n = 3, ± SD).
- Structure Modeling of Est8
A 3D structural model was generated using SWISSMODEL to obtain structural insights into Est8
. The hypothetical YxiM precursor from
(PDB code 2O14_A) belonging to the SGNH hydrolase family with 35% identity to Est8 was used to build the Est8 model. The fidelity of this structure prediction was evaluated using the Procheck program
. The Est8 predicted structure adopted a typical α/β-hydrolase fold, with a central fourstranded parallel twisted β-sheet flanked by eight α-helices (
A). In the Est8 model, the putative catalytic triad Ser11, Asp182, and His185 were positioned within a pocket of the enzyme to serve as the active center, which was the same as the sequence alignment result in
B). The predictions of the catalytic triad further confirmed the results of the site-directed mutagenesis and chemical inhibition studies mentioned previously. However, the mutants of D182A and H185A had less detectable activity than that of S11A, which suggests that D182 and H185 are more critical than S11 for Est8 activity. Therefore, Est8 may use different catalytic mechanisms that have been reported for other SGNH hydrolases.
Predicted 3D structure of Est8. (A) Overview of the whole modeled structure. (B) Representations of the catalytic triad. The proposed active-site residues Ser11, Asp182, and His185 are shown in stick representations. These figures were rendered using SWISS-MODEL and Swiss-Pdb Viewer.
In conclusion, in this study, we characterized the new esterase Est8 that belongs to the SGNH hydrolase subfamily. The enzyme was active toward short-length
-NP esters, with high stability under alkaline pH, a temperature of 50℃, nonionic detergents, denaturant agents, and organic solvents, which are important characteristics required for applications in detergent formulation and biotransformation. The biochemical functions (structure–function relationships) of many SGNH superfamily enzymes remain unknown and require further investigation.
This work was supported by the National Key Technology Support Program (Grant No. 2013BAD10B01) and the National Natural Science Foundation of China (Grant No. 31160229).
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