Identiﬁcation, Characterization, and Preliminary X-ray Diffraction Analysis of a Novel Esterase ( Sc Est) from Staphylococcus chromogenes

: Ester prodrugs can develop novel antibiotics and have potential therapeutic applications against multiple drug-resistant bacteria. The antimicrobial activity of these prodrugs is activated after being cleaved by the esterases produced by the pathogen. Here, novel esterase Sc Est originating from Staphylococcus chromogenes NCTC10530, which causes dairy cow mastitis, was identiﬁed, char-acterized, and analyzed using X-ray crystallography. The gene encoding Sc Est was cloned into the p VFT1S vector and overexpressed in E. coli . The recombinant Sc Est protein was obtained by afﬁnity and size-exclusion puriﬁcation. Sc Est showed substrate preference for the short chain length of acyl derivatives. It was crystallized in an optimized solution composed of 0.25 M ammonium citrate tribasic (pH 7.0) and 20% PEG 3350 at 296 K. A total of 360 X-ray diffraction images were collected at a 1.66 Å resolution. Sc Est crystal belongs to the space group of P2 1 2 1 2 1 with the unit cell parameters of a = 50.23 Å, b = 68.69 Å, c = 71.15 Å, and α = β = γ = 90 ◦ . Structure reﬁnement after molecular replacement is under progress. Further biochemical studies will elucidate the hydrolysis mechanism of Sc Est. Overall, this study is the ﬁrst to report the functional characterization of an esterase from Staphylococcus chromogenes , which is potentially useful in elaborating its hydrolysis mechanism.


Introduction
Multiple drug resistance (MDR) bacteria are an emerging global threat that potentially imposes healthcare and economic issues [1,2]. The production of drug-inactivating enzymes, such as β-lactamase and aminoglycoside modifying enzymes [3], drug elimination from the cell, mutation of an existing target, and acquisition of a target by-pass system have been proposed as major MDR resistance mechanisms. Therefore, the necessity for discovering and developing novel antibiotics with unconventional modes of action has increased in order to overcome these resistance mechanisms [4].
One of the strategies to avoid MDR is antibacterial prodrugs that are pharmacologically inactive and are cleaved by bacterial enzymes to become active antibiotics [5]. Antibacterial prodrugs are synthesized by adding functional groups to the antibiotic skeleton and may have multiple advantages [5]. For example, adding a lipophilic pivaloyloxymethyl to cephalosporin cefditoren increases its absorption in the small intestine [6]. Ester is also a functional group that is added to antibiotics to increase the delivery efficiency, cell permeability, and oral bioavailability of the prodrug [7,8]. Carbenicillin, carfecillin (phenyl ester), and carindacillin (indanyl ester) are some ester-containing antimicrobial prodrugs [9]. Pathogen specificity is another advantage of ester prodrugs. Since such antibacterial prodrugs are transformed by the cytosolic esterase specifically produced by the pathogen, the pathogen is selectively executed [10].
Previously, human esterases were studied for their function in prodrug activation [11]. However, the application of human esterase for antibiotic prodrug activation is limited due to its esterase-dependent localization and expression. Alternatively, analyzing the substrate selectivity and activity of bacterial esterases has provided crucial details for targeting potential antibiotic prodrugs to develop novel antibiotics for the treatment of MDR [5,7,10,12]. Bacterial esterases have a canonical α/β-hydrolase fold that consists of a core β-sheet surrounded by α-helices to catalyze the hydrolysis (EC 3.1.1.X) of a variety of substrates containing ester groups. The esterases use a catalytic triad comprising a nucleophilic serine, a base histidine, and an activating acidic residue (Asp/Glu) to catalyze the hydrolysis of the ester to a carboxylic acid and alcohol. Despite having the same configuration as the enzyme hydrolase and a high degree of sequence homology, esterases have distinct substrate specificities [13][14][15]. Therefore, pathogenic esterases need to be functionally investigated, whereas the biochemical and structural studies may provide valuable information for designing species-specific antimicrobial ester prodrugs. This preliminary study focuses on the substrate specificity and function of esterases derived from pathogens. Herein, we have analyzed the distribution of esterases and lipases across the genome of Staphylococcus chromogenes NCTC10530, the prevalent bacterial pathogen causing dairy cow mastitis. Furthermore, the carboxylesterase annotated as ScEst has been purified, its biochemical properties have been investigated, and preliminary X-ray studies have been conducted.

Phylogenetic Analysis
The subfamily of ScEst was analyzed using a phylogenetic tree based on full-length protein sequences of several lipolytic enzymes that are already classified into specific subfamilies [16][17][18]. A total of 69 protein sequences, including ScEst and other proteins from the S. chromogenes strain NCTC10530 were used for multiple sequence alignment using ClustalX [19]. The neighbor-joining method was used to generate a phylogenetic tree using the MEGA-X [20].

Gene Cloning, Expression, and Purification of Recombinant ScEst Protein
The gene encoding ScEst (GenBank ID: SUM13810) was amplified by PCR and cloned into the pVFT1S plasmid between the BamHI and XhoI restriction sites. The cloned sequence was verified using Sanger sequencing using T7 promoter and terminator primers. E. coli BL21 (λDE3) was transformed with the recombinant plasmid harboring N-terminal 6xHis-tagged ScEst for protein overexpression (Table 1). A single colony from the Luria Bertani (LB) agar plate containing kanamycin was inoculated as a seed culture and grown overnight. The seed culture (20 mL) was inoculated into 1 L of culture medium and kanamycin (50 µg mL −1 ) and incubated at 37 • C at 150 rpm. When the OD 600 of the culture reached 0.4, protein overexpression was induced by adding 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were further incubated at 37 • C for 4 h, and harvested by centrifugation at 6000× g. The cell pellets were resuspended in a lysis buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, and 20 mM imidazole) and disrupted by sonication (Vibra-Cell™, Sonics & Materials, Inc., Danbury, CT, USA) for 30 min at 35% amplitude (on for 2 s and off for 4 s). The soluble fraction of protein was separated by centrifugation at 20,000× g for 40 min.
Recombinant ScEst was purified via a two-step purification process. First, the His-tagbased purification was performed using a His-trap™ FF column (GE Healthcare, Chicago, IL, USA). The supernatant containing the recombinant ScEst was loaded onto the column, and the resin was washed with 10 column volumes of washing buffer. The remaining recombinant ScEst was eluted with two column volume elution buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 300 mM imidazole). The elute was then concentrated to 5 mL and treated with thrombin for three days at 4 • C in a rotating incubator to cleave the His-tag. For the second purification, HiPrep™ Sephacryl ® S-200 HR (Cytiva, Marlborough, MA, USA) connected to an ÄKTA™ Start chromatography system (GE Life Sciences, Piscataway, NJ, USA) was equilibrated with a buffer composed of 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1 mM EDTA, and the protein sample was loaded onto the column. The column was calibrated using cytochrome C (12.4 kDa), carbonic anhydrase (29 kDa), alcohol dehydrogenase (150 kDa), and β-amylase (200 kDa). K av was calculated by (V s − V o )/(V t − V o ), where vs. = elution volume, V o = column void volume, V t = column volume. The purity and concentration of the recombinant ScEst were validated using SDS-PAGE and the Bradford protein assay, respectively.

Enzymatic Analysis
The substrate specificities of ScEst were measured using various p-nitrophenyl esters, including p-nitrophenyl acetate (pNP-C 2 ), p-nitrophenyl butyrate (pNP-C 4 ), p-nitrophenyl hexanoate (pNP-C 6 ), p-nitrophenyl octanoate (pNP-C 8 ), and p-nitrophenyl decanoate (pNP-C 4 ), obtained from Sigma-Aldrich (St. Louis, MO, USA). The esterase activity with acyl carbon chains of various lengths was evaluated by monitoring the p-nitrophenol (pNP) in the solution spectrophotometrically [21]. Storage buffer (1 mL) containing 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1 µg ScEst was prepared, and the reaction was initiated by mixing an equal volume of the substrate (final 0.12 µM). The final concentration of acetonitrile in the reaction mixture kept to 5% to avoid micelle formation of substrates with longer acyl chains. The enzyme reactions were analyzed at 405 nm using an Epoch™ 2 microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA), using the storage buffer as control. Three independent measurements were used to represent the activity data.

Crystallization, Data Collection, and Structural Analysis
Commercially available crystallization solutions, MCSG I-IV (Anatrace Inc., Maumee, OH, USA), and JCSG™ and PGA Screen™ (Molecular Dimensions Inc., Altamonte Springs, FL, USA) were used to screen the crystallization conditions of ScEst. The sitting-drop vapor diffusion method was set up by mixing 300 nL of solution and an equal volume of protein (25 mg mL −1 ) against 80 µL of solution in the reservoir using a mosquito ® liquid-handling robot (TTP Labtech Ltd., Hertfordshire, UK). Subsequently, multiple optimizations using 24-well plates were further carried out to obtain a decent size and quality of crystals. The crystallization data are presented in Table 2.
The single crystal of ScEst was cryoprotected using a mixture of crystallization solution where the crystal of ScEst grew and glycerol (25% w/v) to prevent the crystal from being frozen under a liquid nitrogen stream. The crystal was then mounted on a sample holder. A total of 360 diffraction images were collected at the synchrotron Beamline 7A of the Pohang Accelerator Laboratory (PAL, Pohang-si, Korea) by rotating at 1 • oscillation per frame. The dataset was indexed, integrated, and scaled using the HKL-2000 software package (HKL Research Inc., Charlottesville, VA, USA). The phase of the ScEst structure was successfully determined using the carboxylesterase Est30 (PDB code: 1TQH) with the molecular replacement method. The X-ray diffraction results are listed in Table 3.

Lipolytic Enzymes of S. chromogenes NCTC10530 and Classification of ScEst
Initially, the bacterial esterases and lipases were classified into eight families (I-VIII) and six subfamilies, all of which belong to Family I, based on the biochemical properties and sequence similarity known as the gold standard classification [17]. Recently, several newly identified lipolytic enzymes have been incorporated into the classification system, resulting in its expansion to 35 families and 11 lipase subfamilies [18].
In this study, a total of 27 putative lipolytic enzymes were identified from the in silico analysis of the genome sequence of S. chromogenes strain NCTC10530. These enzyme sequences were aligned with the categorized enzymes ( Figure 1). Among the putative lipolytic enzymes, ScEst was found to be homologous to Family XIII, specifically with thermostable carboxylesterase Est30 from Geobacillus stearothermophilus (AAN81911, 62.30% identity), EstOF4 from Bacillus sp. (AGK06467, 56.50% identity), and EstB2 from Bacillus sp. (AAT65181, 58.54% identity).
Multiple sequence alignment revealed that the active site of ScEst shares a consensus sequence G-X-S-X-G, characteristic of the esterase/lipase family ( Figure 2). ScEst displayed high sequence similarity with the Family XIII proteins. However, a unique region was also identified in ScEst. The amino acid sequence 103-SLNRD-107 follows the active loop in ScEst in contrast to its orthologs, which have GYTVLP in the corresponding region ( Figure 2). Since this site is in the vicinity of the active site, ScEst may have different specificities for substrate recognition or activity. Overall, the phylogenetic and sequence analyses confirmed that ScEst belongs to the XIII family but harbors a unique sequence, which may lead to a distinctive function.

Biochemical Characterization of ScEst
To confirm the esterase activity, ScEst was expressed and purified using a two-step purification process. His-Tag-affinity purification followed by size-exclusion chromatography yielded the recombinant ScEst protein with high purity (>95%), and a molecular weight similar to the calculated molecular weight of 29.2 kDa ( Figure 3A). The molecular weight of ScEst estimated by size-exclusion chromatography on FPLC was consistent with the anticipated size of the dimer ( Figure 3B). The esterase activity of ScEst assessed using p-nitrophenyl esters (p-NP) indicated that ScEst has a substrate preference for acyl derivatives with a short chain length, and the activity declined as the size of the acyl hydrocarbon chain of the substrates increased. When the activity of ScEst against p-nitrophenyl acetate (C2) was considered 100%, the relative activity was approximately 50% and 20% against p-nitrophenyl butyrate (C4) and p-nitrophenyl hexanoate (C6), respectively. Substrates longer than hexanoate did not show any measurable activity.

X-ray Crystallographic Study of ScEst
To determine the three-dimensional structure of ScEst, crystallization screening using more than 1600 conditions, X-ray diffraction experiments, and initial model building were performed. After multiple crystallization refinements, the best single crystal was obtained with 0.25 M ammonium citrate (pH 7.0) and 20% (w/v) PEG 3350 ( Figure 4A). The single crystal was cryoprotected by a brief soaking in 25% glycerol-based cryoprotectant solution and mounted under a liquid nitrogen stream at 100 K. The full coverage of 360 diffraction images was obtained at the highest resolution of 1.66 Å (Figure 4b). The space group of the ScEst crystal belonged to P2 1 2 1 2 1 with the following unit cell parameters: a = 50.23 Å, b = 68.69 Å, c = 71.15 Å and α, β, γ = 90 • . The initial structure of ScEst was generated by molecular replacement using the CCP4i software suite [22]. Thermophilic carboxylesterase Est30 from Geobacillus stearothermophilus (PDB code, 1TQH) showed a high amino acid sequence similarity (61.79% identity) with ScEst, and was thus used as reference [23]. Model building and iterative structure refinement are currently underway using Coot software [24] and Refmac5 [25] in the CCP4i suite.  Full-length protein sequences of 27 putative lipolytic enzymes from the Staphylococcus chromogenes strain NCTC10530 were aligned with bacterial lipolytic enzyme sequences of known categories using multiple sequence alignment (69 sequences). MEGA-X was used to create the phylogenetic tree using the neighbor-joining method. All unclear locations were deleted (using the pairwise deletion option). The percentage of duplicate trees in which the related taxa were clustered together in the bootstrap test (500 repetitions) appears next to each node. The GenBank accession numbers are indicated in parentheses.
bootstrap test (500 repetitions) appears next to each node. The GenBank accession numbers are indicated in parentheses.

Figure 2.
Multiple sequence alignment of ScEst with other esterases of Family VIII. The sequences including that of thermostable carboxylesterase Est30 from Geobacillus stearothermophilus (GenBank AAN81911), EstOF4 from Bacillus pseudofirmus (GenBank AGK06467), and EstB2 from Bacillus sp. 01-855 (Genbank AAT65181) belonging to the bacterial lipolytic enzyme Family VIII were aligned using ClustalX. The conserved sites are highlighted in a darker color, whereas varied or polymorphic sites are shown in a lighter color. The secondary structure deduced from the Est30 structure (PDB code 1TQH) is displayed on the top of the aligned sequences. The conserved sequence at the active site characteristic of Family VIII is indicated with a red bar. The adjacent region specific to the ScEst is marked with a cyan bar. The conserved catalytic triads are indicated with triangles.

Biochemical Characterization of ScEst
To confirm the esterase activity, ScEst was expressed and purified using a two-step purification process. His-Tag-affinity purification followed by size-exclusion chromatography yielded the recombinant ScEst protein with high purity (>95%), and a molecular weight similar to the calculated molecular weight of 29.2 kDa ( Figure 3A). The molecular weight of ScEst estimated by size-exclusion chromatography on FPLC was consistent with the anticipated size of the dimer ( Figure 3B). The esterase activity of ScEst assessed using p-nitrophenyl esters (p-NP) indicated that ScEst has a substrate preference for acyl derivatives with a short chain length, and the activity declined as the size of the acyl hydrocarbon chain of the substrates increased. When the activity of ScEst against p-nitrophenyl acetate (C2) was considered 100%, the relative activity was approximately 50% and 20% against p-nitrophenyl butyrate (C4) and p-nitrophenyl hexanoate (C6), respectively. Substrates longer than hexanoate did not show any measurable activity.

X-ray Crystallographic Study of ScEst
To determine the three-dimensional structure of ScEst, crystallization screening using more than 1600 conditions, X-ray diffraction experiments, and initial model building were performed. After multiple crystallization refinements, the best single crystal was obtained with 0.25 M ammonium citrate (pH 7.0) and 20% (w/v) PEG 3350 ( Figure 4A). The single crystal was cryoprotected by a brief soaking in 25% glycerol-based cryoprotectant solution and mounted under a liquid nitrogen stream at 100 K. The full coverage of 360

Conclusions
The biochemical characteristics of a carboxylesterase ScEst, derived from S. chromogenes NCTC10530, which is the most common bacterial pathogen causing infectious diseases in dairy cows, were examined. The ScEst gene was identified, isolated, overexpressed in E. coli, and the protein was purified with affinity columns and size-exclusion chromatography. The ScEst enzyme prefers the acyl derivatives with a short chain length as substrates. A preliminary crystallographic investigation of ScEst resulted in a high-resolution dataset. We anticipate that elaborating the structure-based enzymatic mechanism of ScEst will provide valuable information for understanding pathogenic esterases and designing ester prodrugs to treat MDR bacteria.

Conclusions
The biochemical characteristics of a carboxylesterase ScEst, derived from S. chromogenes NCTC10530, which is the most common bacterial pathogen causing infectious diseases in dairy cows, were examined. The ScEst gene was identified, isolated, overexpressed in E. coli, and the protein was purified with affinity columns and size-exclusion chromatography. The ScEst enzyme prefers the acyl derivatives with a short chain length as substrates. A preliminary crystallographic investigation of ScEst resulted in a high-resolution dataset. We anticipate that elaborating the structure-based enzymatic mechanism of ScEst will provide valuable information for understanding pathogenic esterases and designing ester prodrugs to treat MDR bacteria.