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. 2017 Aug 31;83(18):e01286-17. doi: 10.1128/AEM.01286-17

Structural and Mechanistic Insights into the Improvement of the Halotolerance of a Marine Microbial Esterase by Increasing Intra- and Interdomain Hydrophobic Interactions

Ping-Yi Li a, Yi Zhang a, Bin-Bin Xie a,✉, Yan-Qi Zhang a, Jie Hao a, Yue Wang a, Peng Wang a, Chun-Yang Li a, Qi-Long Qin a, Xi-Ying Zhang a, Hai-Nan Su a, Mei Shi a, Yu-Zhong Zhang a,b, Xiu-Lan Chen a,✉
Editor: Ning-Yi Zhouc
PMCID: PMC5583480  PMID: 28733281

ABSTRACT

Halotolerant enzymes are beneficial for industrial processes requiring high salt concentrations and low water activity. Most halophilic proteins are evolved to have reduced hydrophobic interactions on the surface and in the hydrophobic cores for their haloadaptation. However, in this study, we improved the halotolerance of a thermolabile esterase, E40, by increasing intraprotein hydrophobic interactions. E40 was quite unstable in buffers containing more than 0.3 M NaCl, and its kcat and substrate affinity were both significantly reduced in 0.5 M NaCl. By introducing hydrophobic residues in loop 1 of the CAP domain and/or α7 of the catalytic domain in E40, we obtained several mutants with improved halotolerance, and the M3 S202W I203F mutant was the most halotolerant. (“M3” represents a mutation in loop 1 of the CAP domain in which residues R22-K23-T24 of E40 are replaced by residues Y22-K23-H24-L25-S26 of Est2.) Then we solved the crystal structures of the S202W I203F and M3 S202W I203F mutants to reveal the structural basis for their improved halotolerance. Structural analysis revealed that the introduction of hydrophobic residues W202 and F203 in α7 significantly improved E40 halotolerance by strengthening intradomain hydrophobic interactions of F203 with W202 and other residues in the catalytic domain. By further introducing hydrophobic residues in loop 1, the M3 S202W I203F mutant became more rigid and halotolerant due to the formation of additional interdomain hydrophobic interactions between the introduced Y22 in loop 1 and W204 in α7. These results indicate that increasing intraprotein hydrophobic interactions is also a way to improve the halotolerance of enzymes with industrial potential under high-salt conditions.

IMPORTANCE Esterases and lipases for industrial application are often subjected to harsh conditions such as high salt concentrations, low water activity, and the presence of organic solvents. However, reports on halotolerant esterases and lipases are limited, and the underlying mechanism for their halotolerance is still unclear due to the lack of structures. In this study, we focused on the improvement of the halotolerance of a salt-sensitive esterase, E40, and the underlying mechanism. The halotolerance of E40 was significantly improved by introducing hydrophobic residues. Comparative structural analysis of E40 and its halotolerant mutants revealed that increased intraprotein hydrophobic interactions make these mutants more rigid and more stable than the wild type against high concentrations of salts. This study shows a new way to improve enzyme halotolerance, which is helpful for protein engineering of salt-sensitive enzymes.

KEYWORDS: esterase, halotolerance, crystal structure, hydrophobic interactions, protein rigidity

INTRODUCTION

Lipolytic enzymes, including esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), catalyze the hydrolysis and synthesis of ester substrates and widely exist in archaea, bacteria, plants, and animals. Esterases act on esters with short acyl chains (≤C10), whereas lipases prefer long-chain esters (≥C10) (1). Esterases and lipases represent two groups of α/β hydrolases. Many lipolytic enzymes do not require cofactors and show broad substrate specificity, high stereoselectivity, and nonaqueous catalytic properties (2). These properties have made esterases and lipases widely used in food, dairy, detergent, paper processing, and cosmetics and the pharmaceutical and agrochemical industries (1, 3). In these industrial processes, lipolytic enzymes are often subjected to harsh conditions, such as high temperatures, high salt contents, and the presence of organic solvents (4, 5). Therefore, it would be of great importance to obtain thermostable, halotolerant, and/or organic solvent-tolerant esterases and lipases for industrial application.

In contrast to the extensive study of thermophilic lipolytic enzymes (6–8), only a few lipolytic enzymes are reported to be halotolerant or halophilic, including a halophilic esterase (HmEST) from Haloarcula marismortui (9, 10), three halotolerant esterases (EstKT4, EstKT7, and EstKT9) from a tidal flat sediment (11), a halotolerant esterase (PE10) from Pelagibacterium halotolerans (12), and a halophilic lipase (LipBL) from Marinobacter lipolyticus (13). Modeled structures of haolophilic HmEST (9) and halotolerant PE10 (12) showed that their protein surface is populated with negatively charged acidic amino acid residues, which may assist in solvation and stability of the enzyme under high-salt conditions. However, due to the lack of structure, the molecular basis for the halotolerance of halotolerant or halophilic lipolytic enzymes is still unclear.

Although structures from halotolerant or halophilic lipolytic enzymes are scarce, structures of other halophilic proteins have been solved, including a malate dehydrogenase from H. marismortui (14), a 2Fe-2S ferredoxin from H. marismortui (15), an endonuclease from Vibrio salmonicida (16), and an alkaline phosphatase from Halobacterium salinarum (17). Halophilic enzymes usually have more acidic residues (Asp and Glu) and less Lys in the sequence, leading to relatively low pI values ranging from 4.3 to 6.8 (18–20). Comparative structural analyses demonstrated that, while halophilic and nonhalophilic proteins are similar in overall structure, secondary structure content, and the number of residues involved in the formation of hydrogen bonds, halophilic proteins have a lower content of nonpolar residues and a larger number of acidic residues distributed on the protein surface, which is supposed to be a key determinant for their haloadaptation (15-17, 21). The negatively charged surface of halophilic proteins tends to bind hydrated cations and forms a solvation shell to maintain the protein stability under high-salt conditions (22–24). Nevertheless, an analysis of the electrostatics of two halophilic proteins suggested that repulsive electrostatic interactions between acidic residues are a major destabilization factor for halophilic proteins to prevent protein aggregation under low/high-salt conditions (25). By removal of surface acidic residues, halophilic proteins could become less halophilic and vice versa (26–28).

Besides the surface electrostatic interactions, the hydrophobic interactions of halophilic proteins have been evolved for haloadaption. Compared to nonhalophilic homologs, halophilic proteins contain a decreased number of large hydrophobic amino acid residues and have reduced hydrophobic interactions on the surface and in the hydrophobic cores (29, 30). Weakening of these interactions may prevent the protein from aggregation and/or inactivation in hypersaline environments (30, 31). In addition, disulfide bonds (32) and interior salt bridges (33) may also contribute to the haloadaption of halophilic proteins.

Marine esterases play an important role in marine organic carbon degradation and cycling. We previously identified a thermolabile esterase, E40, from a surface sediment sample (154-m water depth) from the South China Sea (34, 35). This marine sediment sample contains ∼3.4% (wt/vol) salts, and its temperature is 17.3°C (36). E40 is a cold-adapted enzyme, unstable at temperatures over 30°C but stable at 20°C or below (34), reflecting the adaptation of the thermolabile E40 to the permanently cold marine environment it inhabits.

E40 is a typical esterase from the hormone-sensitive lipase (HSL) family, containing a CAP domain and a catalytic domain (34). Comparative structural analyses of E40 and other thermolabile and thermostable HSLs revealed that the absence of interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain leads to the thermolability of E40 (34). To improve the thermostability of E40, we generated several mutants by introducing hydrophobic residues in loop 1 and/or α7 of E40. Among them, the M3 (defined below) S202W I203F mutant with introduced hydrophobic residues in both loop 1 and α7 is the most thermostable (34). Here, we compared the halotolerance of E40 and its mutants and found that most of the mutants had higher halotolerance than wild-type (WT) E40 and that the M3 S202W I203F mutant was the most halotolerant. Then we solved the crystal structures of two S202W I203F and M3 S202W I203F halotolerant mutants. Structural analyses revealed that E40 mutants become more rigid and more stable than the wild type under high-salt conditions due to the increased intradomain and interdomain hydrophobic interactions, which suggests another way to improve the halotolerance of enzymes.

RESULTS AND DISCUSSION

Effect of NaCl on the activity of E40 and its mutants with introduced hydrophobic residues.

E40 is a thermolabile esterase from a surface sediment sample from the South China Sea (34, 35). By introducing hydrophobic residues in loop 1 and/or α7 of E40, we have obtained several mutants with improved thermostability following the order M3 S202W I203F > S202W I203F > I203F > S202W > M3 ≈ E40 (34). In these mutants, mutation M3 occurs in loop 1 of the CAP domain, in which residues R22-K23-T24 of E40 are replaced by residues Y22-K23-H24-L25-S26 of Est2 to introduce hydrophobic residues Tyr and Leu in loop 1, and mutations S202W and I203F occur in α7 of the catalytic domain (34). Both E40 and its mutants form tetramers in solution (34).

In the process of protein purification, ion-exchange chromatography was applied and WT E40 and its mutants were eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 0.2 to 0.4 M NaCl. We found that WT E40 tended to be inactivated and precipitated in the buffer containing more than 0.3 M NaCl, but its thermostable mutants were not, implying that these mutants are likely more halotolerant than the wild type. Then we investigated the effect of NaCl on the activities of WT E40 and its mutants (Fig. 1). Under their respective optimum temperatures, the activities of both E40 and its mutants decreased upon exposure to increased concentrations of NaCl ranging from 0 to 4.0 M (Fig. 1A). However, the degrees of activity decrease were quite different among them, in the order of WT E40 ≈ I203F mutant > S202W I203F mutant > M3 S202W I203F mutant, suggesting that the M3 S202W I203F mutant with introduced hydrophobic residues in both loop 1 and α7 was the most resistant protein against high-salt conditions (Fig. 1A). E40 had an optimum temperature of 45°C, and all mutants of E40 in this study had optimum temperatures higher than 45°C. Thus, the effects of NaCl on the activities of E40 mutants were also measured at 45°C and compared to that of WT E40. At 45°C, the halotolerance of these proteins followed this order: M3 S202W I203F mutant > S202W I203F mutant > I203F mutant > E40 (Fig. 1B). In addition, at 20°C, close to the sampling temperature of 17.3°C (36), E40 showed stimulated activity (129%) in 0.5 M NaCl, 100% activity in 2.0 M NaCl, and 17.3% activity in 3.5 M NaCl, reflecting the adaption of E40 to the marine saline environment. However, the I203F, S202W I203F, and M3 S202W I203F mutants showed nearly 100% activity in 3.0 M NaCl and more than 87% activities in 3.5 M NaCl (Fig. 1C), also indicating that E40 mutants have improved halotolerance.

FIG 1.

FIG 1

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Effect of NaCl on the activities of E40 and its mutants measured at different temperatures. (A) Effect of NaCl on the activities of E40 and its mutants measured at their respective optimum temperatures. The optimum temperatures for E40 and the I203F, S202W I203F, and M3 S202W I203F mutants were 45, 60, 60, and 55°C, respectively (34). The activities of E40 (240.9 U/mg) and the I203F (450.7 U/mg), S202W I203F (495.8 U/mg), and M3 S202W I203F (378.4 U/mg) mutants in 0 M NaCl were taken as 100%, respectively. (B) Effect of NaCl on the activities of E40 and its mutants measured at 45°C. The activities of E40 (240.9 U/mg) and the I203F (250.1 U/mg), S202W I203F (192.0 U/mg), and M3 S202W I203F (213.7 U/mg) mutants in 0 M NaCl were taken as 100%, respectively. (C) Effect of NaCl on the activities of E40 and its mutants measured at 20°C. The activities of E40 (52.4 U/mg) and the I203F (55.5 U/mg), S202W I203F (76.2 U/mg), and M3 S202W I203F (103.3 U/mg) mutants in 0 M NaCl were taken as 100%, respectively. The graphs show data from triplicate experiments (mean ± standard deviation [SD]).

Effect of NaCl on the stability of E40 and its mutants with introduced hydrophobic residues.

We also compared the stabilities of WT E40 and its mutants in different concentrations of NaCl (Fig. 2). When incubated in 0.5 M NaCl at 20°C, E40 lost all of its activity after 25 min (Fig. 2A), whereas the I203F, S202W I203F, and M3 S202W I203F mutants kept nearly 100% activities after 1 h. Moreover, the I203F, S202W I203F, and M3 S202W I203F mutants still retained 31.1, 40.5, and 68.0% of their activities, respectively, after incubation in 3.0 M NaCl for 1 h (Fig. 2B). These results further indicate that E40 mutants have improved halotolerance and that the M3 S202W I203F mutant is the most stable protein in the presence of high concentrations of NaCl.

FIG 2.

FIG 2

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Effect of NaCl on the stability of E40 and its mutants. (A) Effect of 0.5 M NaCl on the stability of E40. E40 was incubated with 0.5 M NaCl at 20°C for different periods, and the residual activity was assayed at 45°C. The activity in 0 M NaCl (239.2 U/mg) was taken as 100%. (B) Effect of NaCl of different concentrations on the stability of E40 and its mutants. E40 and its mutants were incubated with NaCl at different concentrations for 1 h at 20°C, and their residual activities were assayed at their respective optimum temperatures. The activities of E40 (228.4 U/mg) and the I203F (444.6 U/mg), S202W I203F (503.4 U/mg), and M3 S202W I203F (385.0 U/mg) mutants in 0 M NaCl were taken as 100%, respectively. The graphs show data from triplicate experiments (mean ± SD).

Effect of NaCl on the structures of E40 and its halotolerant M3 S202W I203F mutant.

In the presence of 0.5 M NaCl, there were no significant differences between the circular dichroism (CD) spectra of E40 and its M3 S202W I203F mutant (Fig. 3A and B). After 1 h of incubation in 0.5 M NaCl at 20°C, the circular dichroism (CD) spectra of E40 and its M3 S202W I203F mutant remained unchanged, suggesting that their secondary structures were not affected by 0.5 M NaCl. Moreover, the fluorescence spectra of E40 and its M3 S202W I203F mutant were also unaffected by 0.5 M NaCl (Fig. 3C), suggesting that their tertiary structures were not affected by 0.5 M NaCl. However, gel filtration analysis showed that after 1 h of incubation in 0.5 M NaCl at 20°C, parts of the E40 tetramers were depolymerized, whereas the M3 S202W I203F mutant remained a tetramer (Fig. 3D), indicating that the quaternary structure of E40 was less stable than that of the M3 S202W I203F mutant in 0.5 M NaCl.

FIG 3.

FIG 3

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Effect of NaCl on the structures of E40 and its halotolerant M3 S202W I203F mutant. (A) Far-UV CD spectra of E40 after incubation at 20°C in 0.5 M NaCl for different periods. The protein concentration used for E40 was 0.2 to 0.3 mg/ml. (B) Far-UV CD spectra of the M3 S202W I203F mutant after incubation at 20°C in 0.5 M NaCl for different periods. The protein concentration used for the M3 S202W I203F mutant was 0.2 to 0.3 mg/ml. (C) Fluorescence spectra of E40 and its M3 S202W I203F mutant after incubation at 20°C in 0.5 M NaCl for different periods. The protein concentration used for E40 and the M3 S202W I203F mutant was 0.06 mg/ml. (D) Gel filtration analysis of E40 and its M3 S202W I203F mutant after incubation at 20°C in 0.5 M NaCl for different periods. Gel filtration analysis was carried out on a Superdex 200 column (GE Healthcare) at 4°C using 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl as the running buffer. The protein concentration used for E40 and the M3 S202W I203F mutant was 0.8 mg/ml.

The protein concentration used for CD and fluorescence spectrum analysis was relatively low (<0.3 mg/ml). However, when the protein concentration was increased to 2 mg/ml in 0.5 M NaCl, E40 was inactivated and precipitated in 2 h, whereas the M3 S202W I203F mutant was not, which also suggested that the protein structure of the M3 S202W I203F mutant is more rigid than that of the wild type.

Crystallization and structure solution of the halotolerant S202W I203F and M3 S202W I203F mutants.

Using the hanging-drop vapor diffusion method, crystals of WT E40 in complex with or without phenylmethylsulfonyl fluoride (PMSF) inhibitors grew in the same crystallization buffer. Crystals of WT E40 without PMSF retained high activity, indicating that the hanging-drop vapor diffusion method has little impact on the protein structure of WT E40. Compared to WT E40, the halotolerance of its S202W I203F and M3 S202W I203F mutants was significantly improved. Therefore, the hanging-drop vapor diffusion method is also fit for studying the three-dimensional (3D) structures of E40 mutants.

To analyze the molecular mechanism for the improvement of their halotolerance, the S202W I203F and M3 S202W I203F mutant proteins were crystallized at 20°C using the hanging-drop vapor diffusion method, and their structures were solved by molecular replacement using the E40 structure (PDB code 4XVC) as the starting model. The 1.70-Å resolution crystal structure of the S202W I203F mutant was determined in space group C2221 with two molecules per asymmetric unit, and refinement of this structure finally converged with an Rwork value of 15.9% and an Rfree value of 17.8%. The 1.80-Å-resolution crystal structure of the M3 S202W I203F protein was determined in space group P1 with four molecules per asymmetric unit, and the final refined structure has an Rwork of 17.2% and an Rfree of 20.7%. Although E40 and the S202W I203F and M3 S202W I203F mutants had different oligomerization states in crystal, previous gel filtration analysis demonstrated that they all form tetramers in solution and that the mutations S202W I203F and M3 S202W I203F have no impact on E40 oligomerization in solution (34). The crystallographic statistics for data collection and refinement are summarized in Table 1.

TABLE 1.

Diffraction data and refinement statistics of the S202W I203F and M3 S202W I203F mutants

Parameter Value fora:
S202W I203F mutant M3 S202W I203F mutant
Diffraction data
    Space group C2221 P1
    Unit cell dimensions
        a, b, c (Å) 90.11, 144.84, 96.07 63.13, 73.68, 85.55
        α, β, γ (°) 90.00, 90.00, 90.00 67.54, 68.32, 76.86
    Resolution range (Å) 36.21–1.70 (1.76–1.70) 40.01–1.80 (1.86–1.80)
    Redundancy 13.9 (14.3) 1.9 (1.9)
    Completeness (%) 99.8 (100.0) 96.6 (92.6)
    Rmerge (%)b 6.7 (36.2) 7.1 (31.7)
Refinement statistics
    Rwork (%) 15.9 17.2
    Rfree (%) 17.8 20.7
    RMSDc from ideal geometry
        Bond length (Å) 0.010 0.006
        Bond angle (°) 1.54 1.01
    Ramachandran plot (%)
        Favored regions 96.59 97.70
        Allowed regions 3.41 2.30
    No. of outliers 0 0
    Avg B factors (Å2) 26.71 24.89
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a

The values in parentheses refer to data in the highest-resolution shell.

b

Rmerge = ∑hkl∑i|I(hkl)i − 〈I(hkl)〉|/∑hkl∑i〈I(hkl)i〉.

c

RMSD, root mean square deviation.

Structural basis for the improved halotolerance of the S202W I203F and M3 S202W I203F mutants.

Halophilic proteins usually exhibit reduced hydrophobic interactions on the surface and in the hydrophobic cores, which may prevent the protein from aggregation and/or inactivation in hypersaline environments (30, 31). The incorporation of a single aromatic core converted a halophilic β-trefoil protein to be mesophilic (37). However, our results suggested that the improved halotolerance of E40 mutants may be related to the introduced hydrophobic residues. To reveal the impact of introduced hydrophobic residues on the halotolerance of E40, comparative structural analysis was carried out between E40 and its halotolerant S202W I203F and M3 S202W I203F mutants.

Compared to WT E40, no significant changes in charge distribution are observed on the surface of the two mutants. The overall structures of the S202W I203F and M3 S202W I203F mutants are quite similar to that of E40, showing root mean square deviations of 0.16 and 0.13 Å, respectively (Fig. 4). However, the distance between loop 1 of the CAP domain and the catalytic domain in the M3 S202W I203F mutant is smaller (8.4 Å) than those in E40 and the S202W I203F mutant (both 11.3 Å) (Fig. 4), suggesting that the M3 S202W I203F mutant has more interdomain interactions than E40 and the S202W I203F mutant. Protein Interactions Calculator analysis also showed that, at the monomeric level, interdomain interactions between loop 1 and the catalytic domain are absent in either E40 or the S202W I203F mutant but present in the M3 S202W I203F mutant.

FIG 4.

FIG 4

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Superposition of the structures of wild-type protein E40 and its halotolerant S202W I203F and M3 S202W I203F mutants. For E40, the CAP domain is shown in yellow and the catalytic domain in gray. For the S202W I203F mutant, the CAP domain is shown in orange and the catalytic domain in cyan. For the M3 S202W I203F mutant, the CAP domain is shown in magenta and the catalytic domain in green. The minimum distances between the main-chain atoms of loop 1 and α7 are 11.3 Å in both E40 and the S202W I203F mutant and 8.4 Å in the M3 S202W I203F mutant.

Detailed structural analysis of WT E40 showed that there are no interactions between loop 1 and the catalytic domain and that residue I203 forms hydrophobic interactions with V80 and L206 within the catalytic domain (Fig. 5A). In the presence of 0.5 M NaCl and at 45°C, E40 had a reduced kcat by 34% and an increased Km by 1.75-fold (Table 2), suggesting that the protein structure of E40 is not compact and that its active site is easily affected by NaCl. When I203 is replaced by F203 in the S202W I203F mutant, F203 protrudes into the substrate-binding pocket in a similar orientation to I203, and the large side chain of F203 shortens its distances from surrounding residues V80 and I81, which can strengthen the hydrophobic interactions of F203 with V80, I81, and L206 (Fig. 5B). In the S202W I203F mutant, another replacement of hydrophilic S202 by hydrophobic Trp results in the formation of hydrophobic interactions between W202 and F203, further enhancing the hydrophobic interactions between residues W202 and F203 and other residues in the catalytic domain (Fig. 5B). In 0.5 M NaCl and at the optimum temperature (60°C) or 45°C, the Km of the S202W I203F mutant was increased to a similar degree to that of E40, whereas its kcat was nearly unaffected (Table 2), suggesting that this mutant is more rigid than the wild type due to the increase in intradomain hydrophobic interactions.

FIG 5.

FIG 5

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Detailed comparative structural analysis of E40 and its halotolerant S202W I203F and M3 S202W I203F mutants. (A) Hydrophobic interactions surrounding residue I203 in E40. I203 in α7 forms intradomain hydrophobic interactions with residues V80 and L206. The CAP domain is shown in yellow, and the catalytic domain in gray. (B) Hydrophobic interactions surrounding the residues W202 and F203 in the S202W I203F mutant. F203 forms intradomain hydrophobic interactions with residues V80, I81, W202, and L206. The CAP domain is shown in orange and the catalytic domain in cyan. (C) Hydrophobic interactions surrounding the residues W204 and F205 in the M3 S202W I203F mutant. F205 forms intradomain hydrophobic interactions with residues V82, I83, W204, and L208. There are also interdomain hydrophobic interactions between residues W204 in α7 and Y22 in loop 1. The CAP domain is shown in magenta and the catalytic domain in green. (D) Superposition of the structures of E40 and the S202W I203F and M3 S202W I203F mutants involving loop 1 and α7. In panels A, B, and C, the catalytic triads are in ball-and-stick representation and hydrophobic interactions within 5 Å are shown.

TABLE 2.

Effect of NaCl on the kinetic parameters of E40 and its mutantsa

Enzyme and conditiona Temp (°C) Vmax (μmol/min/mg) Km (mM) kcat (s−1) kcat/Km (mmol−1 s−1)b
WT E40 45 511.5 ± 9.8 0.24 ± 0.01 291.6 ± 7.9 1,209.7 (100)
WT E40 + NaCl 45 337.4 ± 12.2 0.42 ± 0.03 192.3 ± 9.8 457.9 (37.9)
S202W I203F mutant 45 372.3 ± 3.7 0.11 ± 0.01 212.2 ± 2.1 1,929.1 (159.5)
S202W I203F mutant + NaCl 45 427.8 ± 11.5 0.19 ± 0.01 243.8 ± 6.6 1,283.3 (106.1)
M3 S202W I203F mutant 45 389.4 ± 17.3 0.11 ± 0.02 222.0 ± 9.9 2,004.2 (165.7)
M3 S202W I203F mutant + NaCl 45 394.4 ± 19.5 0.13 ± 0.01 224.8 ± 11.1 1,728.0 (142.8)
S202W I203F mutant 60 865.9 ± 27.3 0.15 ± 0.01 493.6 ± 22.0 3,268.6 (270.2)
S202W I203F mutant + NaCl 60 778.2 ± 18.1 0.29 ± 0.02 443.6 ± 14.6 1,556.4 (128.7)
M3 S202W I203F mutant 55 591.8 ± 5.5 0.11 ± 0.01 337.3 ± 4.4 2,933.3 (242.5)
M3 S202W I203F mutant + NaCl 55 619.1 ± 17 0.13 ± 0.01 352.9 ± 13.7 2,693.8 (222.7)
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a

To measure the kinetic parameters of E40 and its mutants, reactions were conducted in triplicate in 50 mM Tris-HCl buffer (pH 7.5) or in 50 mM Tris-HCl buffer (pH 7.5) containing 0.5 M NaCl at their respective optimum temperatures using p-NPC4 as substrate over a concentration range of 0.05 to 4.0 mM. The kinetic parameters of E40 mutants were also measured at 45°C. The optimum temperatures for E40 and the S202W I203F and M3 S202W I203F mutants were 45, 60, and 55°C, respectively (34).

b

Values in parentheses are percentages calculated relative to WT E40.

In the M3 S202W I203F mutant, residues R22-K23-T24 are replaced by residues Y22-K23-H24-L25-S26 to introduce two hydrophobic residues, Y22 and L25, into loop 1. Thus, the loop 1 of the M3 S202W I203F mutant contains two more residues than that of WT E40, and residues S202 and I203 in WT E40 are correspondingly changed to W204 and F205 in the M3 S202W I203F mutant, respectively. Similar to the S202W I203F mutant, the M3 S202W I203F mutant has strengthened intradomain hydrophobic interactions between F205 and the residues V82, I83, W204, and L208 within the catalytic domain (Fig. 5C and D). In addition to intradomain hydrophobic interactions, interdomain hydrophobic and aromatic-aromatic interactions form between the introduced residues Y22 in loop 1 and W204 in α7 (Fig. 5C), which make this mutant more compact than the S202W I203F mutant by shortening the distance between loop 1 and the catalytic domain (Fig. 4). At both the optimum temperature (55°C) and 45°C, the M3 S202W I203F mutant had similar kcat and Km in the buffer with or without 0.5 M NaCl (Table 2), indicating that the protein structure of the M3 S202W I203F mutant is more rigid than that of the S202W I203F mutant and more resistant to NaCl. Therefore, by introducing hydrophobic residues in loop 1 and α7, both intradomain and interdomain hydrophobic interactions are reinforced, and thus the M3 S202W I203F protein becomes more rigid and more stable than the wild type and the S202W I203F mutant under low/high-salt concentrations.

Increasing intra- and interdomain hydrophobic interactions is also a way to improve the halotolerance of enzymes.

Many halotolerant or halophilic proteins, including esterases and lipases, are reported to have an increase in acidic residues and a decrease in solvent-exposed hydrophobic residues on their protein surface, and these structural features are important for their adaption to hypersaline environments (21). By introducing surface acidic residues, nonhalophilic proteins could have halophilic characteristics (28). In this study, biochemical analysis showed that E40 mutants with introduced hydrophobic residues have improved halotolerance following this order: M3 S202W I203F mutant > S202W I203F mutant > I203F mutant > WT E40 (Fig. 1 and 2), in line with the order of their thermostability (34). Further structural analysis demonstrated that increased intra- and interdomain hydrophobic interactions make E40 mutants become more rigid and more stable than the wild type against both high-salt conditions and high temperatures, indicating that increasing intraprotein hydrophobic interactions is also a way to improve the halotolerance of enzymes. These results broaden our understanding of the halotolerance mechanisms of enzymes and may be helpful in protein engineering targeting to improve the stability of enzymes with industrial potential against both high salt concentrations and high temperatures.

MATERIALS AND METHODS

Strains and growth conditions.

Escherichia coli DH5α and E. coli BL21(DE3) were used as the hosts for cloning and expression, respectively. Bacteria were grown at 37°C in Luria-Bertani (LB) medium supplemented with 30 μg/ml kanamycin.

Protein expression and purification.

Expression and purification of WT E40 and its mutants, including the I203F, S202W I203F, and M3 S202W I203F proteins, were carried out under the same conditions as described previously (34).

Enzyme assays.

Esterase activity was measured as described by Li et al. (34). The reaction system contained 50 mM Tris-HCl buffer (pH 8.0), 0.02 ml of 10 mM p-nitrophenyl butyrate (p-NPC4) (dissolved in high-performance liquid chromatography [HPLC]-grade 2-propanol), and 0.02 ml of enzyme (0.09 to 0.17 μM, depending on the specific activity of WT E40 or the mutants) in a final volume of 1 ml. After incubation at 45°C for 5 min, the reaction was terminated by an addition of 0.1 ml of 20% (wt/vol) SDS, and then the absorbance of the reaction mixture at 405 nm was measured on a microplate spectrophotometer. Measurements were corrected for background hydrolysis in the absence of enzyme. One unit of enzyme is defined as the amount of enzyme required to liberate 1 μmol of p-nitrophenol per minute. Enzyme kinetic assays were carried out in 50 mM Tris-HCl buffer (pH 7.5) with or without 0.5 M NaCl at different temperatures using p-NPC4 at concentrations from 0.05 to 4.0 mM. Kinetic parameters were calculated by nonlinear regression fit directly to the Michaelis-Menten equation using the Origin8 software.

Effect of NaCl on enzyme activity and stability.

The activity of E40 and its mutants was determined at NaCl concentrations ranging from 0 to 4.0 M and at their respective optimum temperatures. The activity of E40 and its mutants at different salt concentrations was also measured at 20 and 45°C, respectively. The autohydrolysis of p-NPC4 substrates was observed to be lower than 10% at 45°C and lower than 15% at 60°C. Measurements were corrected for background hydrolysis in the absence of enzyme. The stability of E40 and its mutants at different salt concentrations was determined by measuring the residual activity at their respective optimum temperatures after these enzymes were incubated at 20°C for 1 h in different concentrations of NaCl (0 to 4.0 M). The molar extinction coefficients of the p-nitrophenol product in 50 mM Tris-HCl buffer (pH 8.0) containing different NaCl concentrations are similar at 405 nm: 1.41 × 104 M−1 cm−1 in 0 M NaCl, 1.46 × 104 M−1 cm−1 in 0.5 M NaCl, 1.47 × 104 M−1 cm−1 in 1.0 M NaCl, 1.50 × 104 M−1 cm−1 in 2.0 M NaCl, 1.50 × 104 M−1 cm−1 in 3.0 M NaCl, and 1.51 × 104 M−1 cm−1 in 4.0 M NaCl.

CD spectroscopy.

WT E40 and its M3 S202W I203F mutant at a concentration of 0.2 to 0.3 mg/ml were incubated at 20°C in 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl for different periods. The overall secondary structures of E40 and the M3 S202W I203F mutant were investigated at 25°C using a J-810 CD spectropolarimeter (JASCO). CD spectra were collected from 200 to 250 nm at a scanning rate of 200 nm/min with a path length of 0.1 cm.

Fluorescence measurements.

Steady-state fluorescence measurements were performed using a Jasco FP-6500 spectrofluorometer equipped with a Peltier computer-controlled thermostat. The excitation and emission wavelengths were set at 280 and 300 to 450 nm, respectively. Both excitation and emission bandwidths were 5 nm. Cuvettes with a 1-cm path length were used. The protein concentration was 0.06 mg/ml for WT E40 and its M3 S202W I203F mutant. Fluorescence spectra of WT E40 and its M3 S202W I203F mutant after incubation in 0.5 M NaCl for different periods at 20°C were recorded, respectively.

Crystallization and structure solution.

Proteins of the S202W I203F and M3 S202W I203F mutants for crystallization were diluted to 2.0 mg/ml in 10 mM Tris-HCl (pH 8.0) containing 100 mM NaCl, respectively. Crystals suitable for X-ray diffraction were obtained using the hanging-drop vapor diffusion method. Crystals of S202W I203F protein grew in the buffer containing 0.1 M imidazole (pH 7.0) and 50% (vol/vol) 2-methyl-1,3-propanediol (MPD) at 20°C for 1 week. Crystals of M3 S202W I203F protein grew in the buffer containing 0.1 M succinic acid (pH 6.5) and 15% (wt/vol) polyethylene glycol 3350 (PEG 3350) at 20°C for 1 week. X-ray diffraction data were collected on the BL17U1 beamline at the Shanghai Synchrotron Radiation Facility using Area Detector Systems Corporation Quantum 315r. The initial diffraction data sets were processed by the HKL2000 program (38). The crystal structures of the S202W I203F and M3 S202W I203F mutants were solved by molecular replacement using the CCP4 program Phaser (39) with E40 (PDB code 4XVC) as the starting model. Subsequent refinement was performed using Coot (40) and Phenix (41). All structure figures were generated using PyMOL software (http://pymol.sourceforge.net). The Protein Interactions Calculator server (42) was used to analyze the intraprotein interactions of E40 and the S202W I203F and M3 S202W I203F mutants.

Accession number(s).

The structures of the S202W I203F and M3 S202W I203F mutant proteins have been deposited in the Protein Data Bank (PDB) under accession no. 5GMS and 5GMR, respectively.

ACKNOWLEDGMENTS

We thank the staff of BL17U1 Beamline at the National Center for Protein Sciences Shanghai and Shanghai Synchrotron Radiation Facility, Shanghai, People's Republic of China, for assistance during data collection.

This work was supported by the National Natural Science Foundation of China (grants 91228210, 91328208, 31670497, and 41676180), the Program of Shandong for Taishan Scholars (2009TS079), the Qingdao National Laboratory for Marine Science and Technology (QNLM2016ORP0310), and the Young Scholars Program of Shandong University (2016WLJH36 and 2016WLJH41).

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