Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation

    Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation Jerusha Boyineni, Junyoung Kim, Beom Sik Kang, ChangWoo Lee, Sei-Heon Jang PII: DOI: Reference:

S1570-9639(14)00065-X doi: 10.1016/j.bbapap.2014.03.009 BBAPAP 39322

To appear in:

BBA - Proteins and Proteomics

Received date: Revised date: Accepted date:

12 February 2014 10 March 2014 17 March 2014

Please cite this article as: Jerusha Boyineni, Junyoung Kim, Beom Sik Kang, ChangWoo Lee, Sei-Heon Jang, Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation, BBA - Proteins and Proteomics (2014), doi: 10.1016/j.bbapap.2014.03.009

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Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a

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W208Y mutation

Janga,*

Department of Biomedical Science and Center for Bio-Nanomaterials, Daegu University,

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a

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Jerusha Boyinenia, Junyoung Kima, Beom Sik Kangb, ChangWoo Leea, and Sei-Heon

b

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Gyeongsan 712-714, South Korea School of Life Sciences and Biotechnology, Kyungpook National University, Daegu 702-701,

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South Korea

*Corresponding author. Sei-Heon Jang, Department of Biomedical Science, Daegu

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University, Gyeongsan 712-714, South Korea. Tel: +82-53-850-6462; Fax: +82-53-850-6469;

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E-mail address: [email protected]

Abbreviations: pNPA, p-nitrophenyl acetate; WT, wild-type.

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ACCEPTED MANUSCRIPT Abstract

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Hydrophobic interactions are known to play an important role for cold-adaptation of proteins;

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however, the role of amino acid residue, Trp, has not been systematically investigated. The

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extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on

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thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W208 was the most crucial in maintaining structural conformation

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and thermal stability of the enzyme. Surprisingly, mutation of W208 to Tyr (W208Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in

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the activity at 40C compared to wild-type EstK. The structure model of W208Y suggested

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that Y208 could form a hydrogen bond with D308, which is located next to catalytic residue

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H307, stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of

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cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.

Keywords: cold-adapted enzyme; esterase; thermal stability; catalytic site; Pseudomonas mandelii

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ACCEPTED MANUSCRIPT 1. Introduction

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Cold-adapted enzymes display high catalytic activity at low temperatures due to their

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flexible structure, especially around the catalytic domain, the result of decreased

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intramolecular interactions, such as hydrogen bonds and hydrophobic interactions [1]. As a result, cold-adapted enzymes are vulnerable to heat even at ambient temperatures. Reduced

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activation enthalpy (∆H‡) of cold-adapted enzymes lowers the Gibbs free energy of activation (∆G‡) that is responsible for high catalytic activity at low temperatures [2].

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Many attempts have been made to increase the weak thermal stability of cold-adapted enzymes for applications at ambient temperatures. These include substituting stabilizing

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residues from mesophilic homologs using site-directed mutagenesis [3, 4], introducing

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stabilizing interactions [5-7], or immobilization on solid supports [8]. However, introducing

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stabilizing interactions into cold-adapted enzymes leads to a loss of flexibility and kinetic optimization at low temperatures, despite increased thermal stability [5-7]. It is a challenge to introduce mutations that provide thermal stability into cold-adapted enzymes, while

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maintaining their high catalytic activity. Although the active site of cold-adapted enzymes are known to be unstable, only a few attempts have been made to improve their catalytic site thermal stability [9, 10]. The aim of this study is to investigate the role of hydrophobic amino acid residue Trp on cold-adaptation of an extracellular esterase, EstK, isolated from the psychrotrophic bacterium Pseudomonas mandelii. EstK was active at low temperatures and showed substrate preferences for short-chain fatty acids [11, 12]. EstK is a unique cold-adapted enzyme because it has 5 Trp residues (W110, W159, W208, W236 and W238). Interestingly, EstK is homologous to the thermal adapted esterases, EstE1, isolated from thermal environmental 3

ACCEPTED MANUSCRIPT samples [13, 14], and AFEST, isolated from Archaeoglobus fulgidus [15]. EstE1 and AFEST each have 2 Trp residues and are 36% and 30% similar to EstK, respectively. The 2 Trp

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residues of EstK, W159 and W236, are conserved in EstE1 and AFEST. In this study, we

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constructed a recombinant EstK protein without its signal peptide (rEstKp) and its 5 Trp to

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Phe mutants, each with a single Trp being substituted with Phe. Three additional mutants for W208 (W208A, W208K, and W208Y) were also generated. Hence, each mutant rEstKp retains 4

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Trp residues. The effects of Trp mutation on the thermal stability, structural flexibility, and catalytic activity of rEstKp were investigated using fluorescence spectroscopy, circular

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dichroism spectroscopy, thermodynamic analysis, and temperature-dependent inactivation.

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2. Materials and methods

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2.1 Materials

The pET28a expression vector was purchased from Novagen (Madison, WI). HisTrap and

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Capto Q columns were purchased from GE Healthcare (Piscataway, NJ). EZchange sitedirected mutagenesis kit was purchased from Enzynomics (South Korea). p-Nitrophenyl acetate (pNPA) was purchased from Sigma (St. Louis, MO). All other reagents were obtained from Sigma, unless noted otherwise.

2.2 Construction of expression plasmid for wild-type (WT) rEstKp

Based on the nucleotide sequence of the estK gene (GenBank ID JN983948), which was cloned from Pseudomonas mandelii [12], the following primers were designed to generate the 4

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sequence

without

its

signal

sequence:

primer,

forward

5΄-

CCATGGATGGGGTTGAACACAACAC-3΄ (Nco I site underlined and the N-terminal part WT

in

boldface

type),

reverse

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of

primer,

5΄-

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CCCCTCGAGCTTCTTCAGGTGCTGCTTCAG-3΄ (Xho I site underlined and the C-

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terminal part of WT in boldface type). The resulting PCR product was cloned into the pET28a

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expression vector and confirmed by DNA sequence analysis.

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2.3 Site-directed mutagenesis

The cDNAs for Trp mutants were generated using an EZchange site-directed mutagenesis

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kit according to the manufacturer’s instructions. Trp was replaced by Phe to generate 5 Trp

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mutants (W110F, W159F, W208F, W236F, and W238F). Mutation of W208 to Ala, Lys, or Tyr

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generated three additional Trp mutants (W208A, W208K, and W208Y). The primers used for site-directed mutagenesis are as follows:

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W110F 5΄-ggcggaTTCgtgctgggagatttccc-3΄ and 5΄-cagcacGAAtccgccgccgtgg-3΄ W159F 5΄-ccaaaTTCgtggccgagcacg-3΄ and 5΄-gccacGAAtttggtcgcggc-3' W208F 5΄-gctgTTCccggtgaccgatgcc-3΄ and 5΄-caccggGAAcagcagcacctgg-3΄ W236F 5΄-gaagTTCttctgggacaactacac-3΄ and 5΄-cccagaaGAActtcatcatgtttttgg-3΄ W238F 5΄-ggttcTTCgacaactacaccac-3΄ and 5΄-gttgtcGAAgaaccacttcatcatg-3΄ W208A 5΄-gctgGCGccggtgaccgatgcc-3΄ and 5΄-caccggCGCcagcagcacctgg-3΄ W208K 5΄-gctgAAGccggtgaccgatgcc-3΄ and 5΄-caccggCTTcagcagcacctgg-3΄ W208Y 5΄-gctgTACccggtgaccgatgcc-3΄ and 5΄-caccggGTAcagcagcacctgg-3΄

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2.4 Expression and purification of WT and Trp mutants

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The WT and 8 Trp mutants of rEstKp were expressed in E. coli BL21 (DE3) and purified as previously described [12]. Briefly, the soluble proteins from the cell lysates after sonication

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were purified to homogeneity by nickel-chelate affinity column chromatography and Capto Q column chromatography. The 8 Trp mutants showed substrate preferences for short-chain p-

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2.5 Fluorescence spectroscopy

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liquid nitrogen and stored at –80°C.

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nitrophenyl esters, especially pNPA (data not shown). The purified enzymes were frozen in

The fluorescence emission spectra of WT and Trp mutants were measured using a Scinco FS-2 fluorescence spectrometer at 25°C. Because we detected no difference in fluorescence

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emission spectra obtained at an excitation wavelength of 295 nm for the WT EstK protein at temperatures from 4 to 80 C, we instead used a wavelength of 280 nm, which resulted in distinguishable responses to changes in temperatures (data not shown). A bandwidth of 4 nm was used for excitation and emission. The fluorescence emission spectra were obtained with 5 µM of the enzyme in Buffer A (20 mM Tris·HCl, pH 8.0, 25 mM KCl, 0.1 mM EDTA, and 5 % glycerol). Data were plotted using a GraphPad Prism software. The process of unfolding induced by heat was recorded by measuring the intrinsic fluorescence of the protein samples in Buffer A, which was incubated at various temperatures for 6 min. Acrylamide-dependent fluorescence quenching of WT and Trp mutants was monitored in 6

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the presence of increasing concentrations of acrylamide (0–0.5 M) mixed with 5 µM of

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enzyme in Buffer A. Quenching data were shown as the ratio of intrinsic fluorescence

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intensity (F0) to the fluorescence intensity with 0.5 M acrylamide (F). F0/F values were

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plotted as a bar diagram.

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2.6 Enzyme assay

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The enzyme activity was determined by measuring the amount of p-nitrophenol formed by hydrolysis of pNPA using 100 pmol of the enzyme. The concentration of p-nitrophenol was measured with a Shimadzu UV-1800 spectrophotometer at 400 nm. One enzyme unit releases

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1 µM p-nitrophenol per min at 25°C from pNPA. The reaction was carried out in 1 ml of Buffer B (100 mM Tris·HCl, pH 7.5, 100 mM NaCl, and 0.3% Triton X-100) at 25C for 3

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min. Finally, the background rate of hydrolysis of pNPA was subtracted.

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2.7 Enzyme kinetics and thermodynamics

Michaelis-Menten constant (Km) and catalytic rate constant (kcat) were calculated from the Lineweaver-Burk plots obtained at various temperatures (4, 10, 20, 30, 40 and 50°C). The kcat values were used to construct an Arrhenius plot (lnkcat versus 1/T). The activation energy (Ea) of pNPA hydrolysis was determined from the slope of this plot. Thermodynamic parameters, Gibbs free energy of activation (∆G‡), enthalpy (∆H‡), and entropy (∆S‡) were calculated at their respective temperatures as described by Lonhienne et al. [2] using the following equations,

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∆H‡ = Ea – RT

(Eq. 2)

∆S‡ = (∆H‡ – ∆G‡)/T

(Eq. 3)

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(Eq. 1)

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∆G‡= RT  [ln(kBT/h) – lnk]

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where kB, h, k, and R represent Boltzmann constant, Plank constant, rate constant at

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temperature T in degrees Kelvin, and gas constant, respectively.

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2.8 Thermal stability

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The remaining enzymatic activities of WT and Trp mutants were measured at the apparent

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optimum temperature (50C) for 3 min in Buffer B after incubation at the indicated temperatures, 2 h intervals for up to 10 h.

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2.9 Circular dichroism (CD) spectroscopy

The CD spectra were measured at the Korea Basic Science Institute (Ochang, South Korea) on a JASCO J-715 spectropolarimeter at 25°C using a quartz cuvette of 0.1 cm path length. The protein sample concentration was 0.38 mg/ml in Buffer A. Prior to the measurement; samples were incubated at the indicated temperatures. The spectra were averages of three scans, and were plotted as ellipticity (mdeg) versus wavelength (nm), using GraphPad Prism software. The α-helical contents of WT and Trp mutants were calculated from the CD spectra using K2D3 [16]. 8

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3. Results and discussion

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3.1 Effect of Trp to Phe mutation on protein fluorescence and secondary structure

To explore the contribution of each Trp residue to the total emission, the fluorescence

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emission spectra of WT and 5 Trp mutants (W110F, W159F, W208F, W236F or W238F) were measured. Interestingly, the fluorescence intensity of W208F (red) and W110F (cyan) were less

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than that of WT (black) by 40% and 12%, respectively. However, the fluorescence intensity of W159F, W236F, and W238F were similar to that of WT (Fig. 1A). Further, W208F showed a blue-

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shift of the maximum emission spectrum at 329 nm compared to that of WT at 377 nm (Fig.

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1A). The results indicate that W208 is the major contributor to the maximum fluorescence of

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EstK, and W208F has a different conformation compared with WT. To investigate the extent of Trp exposure to the aqueous phase, the permeabilities of WT and 5 Trp mutants were accessed using water soluble acrylamide, which can penetrate the

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solvent accessible surfaces of the protein tertiary structure. Fig. 1B shows that W208F exhibits a larger slope in the Stern-Volmer plot in the presence of acrylamide compared to WT and other Trp mutants. The results indicate that the W208F mutation had a larger conformational change than WT as it has greater accessibility to acrylamide. The effect of other Trp mutants (W110, W159, W236 and W238) was similar to WT as demonstrated by the reduced fluorescence intensity shown in Fig. 1A. The hydropathy profile calculated by the algorithm of Kyte and Doolittle [17] also provided evidence that the region surrounding W208 is hydrophobic (data not shown). Further, W208 may form hydrophobic interactions with the surrounding amino acid residues to maintain an intact conformation of EstK. 9

ACCEPTED MANUSCRIPT Next, the effect of the Trp to Phe mutations on the secondary structure of EstK was measured as shown in Fig. 1C. The WT and Trp mutants exhibited a well-defined α-helical

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structure overall at 25°C, but W208F exhibited a distinct CD spectrum with increased -helical

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content (51.2%) compared to that of WT (33.6%) and other Trp mutants (Fig. 1C). The -

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strand content of W208F (8.2%) was lower than that of WT (16.2%). Comparison of the secondary structure with hyper-thermophilic homologs EstE1 and AFEST showed that EstK

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has similar -helical and -strand contents with EstE1 (-helix 39.5%, -strand 16.7%) [14] and AFEST (-helix 43.1%, -strand 21.5%) [15]. Taken together, the data indicate that W208,

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out of the 5 Trp residues, is located in the hydrophobic region of the enzyme and mutations of

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3.2 Thermal stability

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Trp to Phe did not affect its overall secondary structure.

To evaluate the role of each Trp residue with regard to thermal stability, the esterase activities of WT and 5 Trp mutants were measured, following an incubation at various

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temperatures (Fig. 2, upper and middle panels). WT maintained its thermal stability for 10 hours in the temperature range from 4 to 50C (Fig. 2); at 60C, its thermal stability was previously shown to decrease rapidly [11, 12]. Among the 5 Trp mutants, W208F was the most sensitive to heat; its esterase activity rapidly decreased upon incubation at 40°C, even though the aromatic side chains of both Trp and Phe can participate in hydrophobic interactions. In contrast to W208F, the other Trp mutants (W110F, W159F, W236F, and W238F) maintained their thermal stability at 40C (Fig. 2). However, at 50°C all Trp to Phe mutants quickly lost their thermal stability, suggesting that Trp is preferred to Phe at those positions. The results indicate that W208 plays a crucial role in maintaining thermal stability of EstK at its catalytic site at 10

ACCEPTED MANUSCRIPT ambient temperatures. To further investigate the role of amino acid residues at position 208 for thermal stability,

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additional substitutions were generated including Ala (non-polar), Lys (basic), and Tyr (polar,

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uncharged). W208A and W208Y maintained their esterase activities upon incubation at 40C.

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However, W208K quickly lost its esterase activity, as seen in W208F (Fig. 2, lower panel). We speculate that the type of amino acid at position 208 is important as Trp and Tyr are

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significantly more polar than Phe, making a hydrogen bond from the nitrogen of the Trp indole group and the hydroxyl group of Tyr, respectively. The non-polar residue Ala in W208A

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is considered to exert a different effect on the thermal stability due to its small side chain size

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compared with Trp and Tyr.

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3.3 Kinetics and thermodynamic analysis

To elucidate the role of Trp residues in cold-adaptation of EstK, kinetic and thermodynamic analyses were performed for WT and Trp mutants using pNPA as substrates.

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As the cold-adapted enzymes exhibit a more ordered structure at the ES‡ complex compared to ES [1], WT and all Trp mutants showed negative T∆S‡ values; the T∆S‡ of WT were -42.5 kJ/mol at 20°C and -44.8 kJ/mol at 40°C. The enzymes showed a more ordered structure at the ES state at 20C than at 40C (Table 1); The entropy change of W159F, W236F, and W238F was larger than WT (Table 1), suggesting that the mutants exhibit more disordered conformations than WT. On the other hand, the entropy change of W208 mutants (W208A, W208F, and W208Y) was less than WT (Table 1), suggesting that the catalytic domain of the mutants might have become rigid by acquiring intramolecular bonds. The thermodynamic parameters of W208K, however, could not be determined. Furthermore, the W208 mutants 11

ACCEPTED MANUSCRIPT showed an increased enthalpy of activation (∆H‡), which supports the idea that the mutation induces a conformational change in EstK as demonstrated by the decreased fluorescence

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intensity and increased acrylamide quenching of W208F as shown in Fig. 1A.

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Surprisingly, W208Y showed the lowest ∆G‡ values and the highest catalytic activity

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among WT and all Trp mutants. W208Y had a ∆G‡ of 62.0 kJ/mol at 20C while WT had a ∆G‡ of 68.6 kJ/mol (Table 1). The catalytic activity of W208Y was 18-fold higher than that of

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which is usual for cold-adapted enzymes.

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WT at 20C (Table 1). The low ∆G‡ of W208Y is considered to be associated with its high kcat,

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3.4 Changes in secondary structure and conformational flexibility

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To elucidate the changes in the overall secondary structure associated with catalytic

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activity, CD spectra of WT and W208 mutants were measured at 4C and 40°C. The -helical content of WT was increased to 41.7% at 40C compared to 33.6% at 4C. However, the helical content of W208 mutants was decreased at 40C compared to that at 4C. The loss of -

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helical content for W208F and W208K at 40C was particularly notable; 51.2% to 32.5% for W208F and 40.9% to 2.1% for W208K. These data support the observation of the decreased thermal stability of W208F and W208K observed in Fig. 2, and suggest that the conformations of W208F and W208K, which maintain the α-helical content at 4C, are not appropriate for maintaining thermal stability at 40°C. The -helical content of W208Y decreased slightly from 48% at 4C to 36.6% at 40C. To investigate the structural flexibility of WT and W208 mutants, their structural motions at 4C and 30°C were measured using acrylamide quenching as shown in Fig. 4. The difference between quenching reflects a difference in the structural permeability or conformational 12

ACCEPTED MANUSCRIPT flexibility [18]. Interestingly, W208F showed a decreased acrylamide quenching at 30C compared to 4C. Conversely, WT and other W208 mutants showed similar permeabilities at

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4C and 30C (Fig. 4). The data suggest that W208F maintains a flexible conformation at 4C,

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which is consistent with the CD spectrum shown in Fig. 3. However, W208F undergoes a conformational change at 30C, which decreases its flexibility.

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3.5 Structure model of W208Y

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A structural model of EstK was constructed using a Swiss-Model based on the crystal structure of EstE1 (PDB code 2c7b), as shown in Fig. 5A. The catalytic triad of EstK,

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consisting of S180-H307-D277, and W208, is located in the 6-strand adjacent to H307 (Fig. 5B).

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In the structure model of W208Y, a hydrogen bond was suggested to form between Y208 and

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D308; thus stabilizing the loop holding the residue H307 and catalytic site of W208Y (Fig. 5C). This increased catalytic site stability of the cold-adapted enzyme is novel as Tyr is located in the corresponding position in the hyper-thermophilic esterases, EstE1 and AFEST (Fig. 5D).

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In the crystal structure of EstE1, the hydroxyl group of Tyr182 was found to form a hydrogen bond to the NH group of Val284. There have been only a few studies that focused on improving the weak catalytic site thermal stability of the cold-adapted enzymes [9], while the majority of previous studies have focused on increasing the overall stability of protein structure [3-7]. To investigate the catalytic site stability of W208Y as shown in Table 1, the esterase activity of W208Y was measured at its optimum temperature, 50C, after incubation at various temperatures as shown in Fig. 6A. The catalytic site of W208Y was resistant to heat and showed a high catalytic activity from 4C to 50°C with a 13-fold increase in the activity at 4C and approximately 17-fold increase from 20C to 50°C compared to WT. Similarly, 13

ACCEPTED MANUSCRIPT replacing active site residues in a cold-active alkaline phosphatase with those found in its mesophilic esterase from Escherichia coli (D116/K274) lead to an increased global heat stability,

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possibly by formation of a salt bridge; however, all the mutants showed reduced substrate

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affinities and lower overall reaction rates [9].

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To further elucidate the process of temperature-induced unfolding of the secondary and tertiary structures of W208Y associated with its increased catalytic site stability, CD and

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fluorescence spectra of W208Y were measured at various temperatures. Despite high catalytic site stability of W208Y, its overall secondary and tertiary structures were weaker than WT (Fig.

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6B and 6C). The fluorescence emission spectra suggested that W208Y was approximately 80% unfolded at 50C in 6 min, whereas WT was approximately 15% unfolded at 50C (Fig. 6B).

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The CD spectra showed that the secondary structures of WT and W208Y were kept intact at

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60C in 3 min (data not shown). However, the secondary structure of W208Y at 60C was

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denatured in 6 min, while WT was kept intact. The secondary structure of WT was denatured at 70C (Fig. 6C). The data suggest that W208Y maintained its functional active site, despite

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denaturation of the protein.

4. Conclusion

We have demonstrated that among the 5 Trp residues in EstK, W208 plays a unique and crucial role in maintaining thermal stability and structural conformation of the enzyme. The weak catalytic site thermal stability of EstK could be enhanced by mutating W208 to Tyr, whereby a hydrogen bond formation between Y208 and D308 may stabilize the catalytic site. The mutations of Tyr182 in EstE1, and vice versa, were sought to elucidate the relationship between structure and function of cold-adapted EstK with its hyper-thermophilic esterase 14

ACCEPTED MANUSCRIPT homologs, EstE1 and AFEST. Studies on the evolutionary relationship of the cold-adapted and thermal adapted enzymes will widen our understanding the mechanism regarding cold-

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adaptation of enzymes from psychrophilic organisms.

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Acknowledgements

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The authors thank Dr. Judith A. Jaehning and Mark A. Karlok for critical reading of the manuscript, and Jeong Soon Park (Korea Basic Science Institute) for measuring CD

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spectra. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-

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2012R1A1A2042195) (to C.L.).

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ACCEPTED MANUSCRIPT References

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[1] C. Struvay, G. Feller, Optimization to low temperature activity in psychrophilic enzymes,

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Int. J. Mol. Sci., 13 (2012) 11643-11665.

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[2] T. Lonhienne, C. Gerday, G. Feller, Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility, Biochim. Biophys. Acta, 1543 (2000)

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1-10.

[3] G. Yang, C. De Santi, D. de Pascale, S. Pucciarelli, C. Miceli, Characterization of the first

MA

eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the

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homologous protein from the mesophilic Euplotes crassus, Biochimie, 95 (2013) 1795-1806.

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[4] C. De Santi, L. Durante, P.D. Vecchio, M.L. Tutino, E. Parrilli, D. de Pascale, Thermal

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stabilization of psychrophilic enzymes: a case study of the cold-active hormone-sensitive lipase from Psychrobacter sp. TA144, Biotechnol. Prog., 28 (2012) 946-952. [5] S. D'Amico, J.C. Marx, C. Gerday, G. Feller, Activity-stability relationships in

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extremophilic enzymes, J. Biol. Chem., 278 (2003) 7891-7896. [6] K.S. Siddiqui, G. Feller, S. D'Amico, C. Gerday, L. Giaquinto, R. Cavicchioli, The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase, J. Bacteriol., 187 (2005) 6197-6205. [7] A. Cipolla, S. D'Amico, R. Barumandzadeh, A. Matagne, G. Feller, Stepwise adaptations to low temperature as revealed by multiple mutants of psychrophilic alpha-amylase from Antarctic Bacterium, J. Biol. Chem., 286 (2011) 38348-38355. [8] H. Lee, H.K. Jeong, J. Han, H.S. Chung, S.H. Jang, C. Lee, Increased thermal stability of cold-adapted esterase at ambient temperatures by immobilization on graphene oxide, 16

ACCEPTED MANUSCRIPT Bioresour. Technol., 148 (2013) 620-623. [9] K. Gudjonsdottir, B. Asgeirsson, Effects of replacing active site residues in a cold-active

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alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli,

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FEBS J., 275 (2008) 117-127.

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[10] B. Asgeirsson, B.V. Adalbjornsson, G.A. Gylfason, Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase,

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Biochim. Biophys. Acta, 1774 (2007) 679-687.

[11] S. Hong, C. Lee, S.H. Jang, Purification and properties of an extracellular esterase from

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a cold-adapted Pseudomonas mandelii, Biotechnol. Lett., 34 (2012) 1051-1055. [12] C. Lee, J. Kim, S. Hong, B. Goo, S. Lee, S.H. Jang, Cloning, expression, and

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characterization of a recombinant esterase from cold-adapted Pseudomonas mandelii, Appl.

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Biochem. Biotechnol., 169 (2013) 29-40.

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[13] J.K. Rhee, D.Y. Kim, D.G. Ahn, J.H. Yun, S.H. Jang, H.C. Shin, H.S. Cho, J.G. Pan, J.W. Oh, Analysis of the thermostability determinants of hyperthermophilic esterase EstE1 based on its predicted three-dimensional structure, Appl. Environ. Microbiol., 72 (2006) 3021-3025.

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[14] J.S. Byun, J.K. Rhee, N.D. Kim, J. Yoon, D.U. Kim, E. Koh, J.W. Oh, H.S. Cho, Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties, BMC Struct. Biol., 7 (2007) 47. [15] G. De Simone, V. Menchise, G. Manco, L. Mandrich, N. Sorrentino, D. Lang, M. Rossi, C. Pedone, The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus, J. Mol. Biol., 314 (2001) 507-518. [16] C. Louis-Jeune, M.A. Andrade-Navarro, C. Perez-Iratxeta, Prediction of protein secondary structure from circular dichroism using theoretically derived spectra, Proteins, 80 (2011) 374-381. 17

ACCEPTED MANUSCRIPT [17] J. Kyte, R.F. Doolittle, A simple method for displaying the hydropathic character of a protein, J. Mol. Biol., 157 (1982) 105-132.

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[18] M.A. Tang, H. Motoshima, K. Watanabe, Fluorescence studies on the stability, flexibility

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mesophilic bacteria, Protein J., 31 (2012) 337-344.

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and substrate-induced conformational changes of acetate kinases from psychrophilic and

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ACCEPTED MANUSCRIPT Table 1 Kinetic and thermodynamic parameters of WT and Trp mutants. kcat

kcat/Km

(µM)

(s-1)

(s-1µM-1)

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104  0.5

3  0.4

0.03

40

356  29.3

10  0.7

0.02

20

2217  698

41  11

40

2399  1151

33  10

20

158  21.2

4  0.1

40

237  14.8

8  0.1

20

110  6.4

12  1.4

40

177  54.4

20

100  1.4

40

155  24.7

W238F

26.1

-44.8

70.9

0.02

ND

ND

ND

0.01

ND

ND

ND

0.03

24.6

-43.7

68.3

0.03

24.4

-46.9

71.3

0.11

14.5

-51.1

65.6

20  0.1

0.11

14.3

-54.6

68.9

10  0.1

0.10

16.9

-49.2

66.1

0.12

16.7

-52.5

69.2

12  0.1

0.03

51.0

-14.6

65.6

156  18

0.30

50.0

-15.8

66.7

160  12

8  0.4

0.05

60.0

-6.7

66.7

182  6.4

39  0.1

0.20

59.8

-7.4

67.2

W208 mutants 20

20

86  9.2

4  0.1

0.04

ND

ND

ND

40

44  5

6  0.7

0.12

ND

ND

ND

20

214  0

54  2.3

0.25

37.4

-24.6

62.0

40

238  0.7

116  2

0.48

37.1

-27.1

64.1

20

W208Y

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40 W208K

396  5

48  5

40 W208F

16  0.7

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W208A

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W236F

68.8

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W159F

(kJ/mol) (kJ/mol) (kJ/mol) -42.5

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W110F

∆G‡

26.3

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T∆S‡

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(°C)

∆H‡

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Km

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Tm

ND: Not determined. Km and kcat correspond to the mean  S.D. for three experiments.

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ACCEPTED MANUSCRIPT Figure legends

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Fig. 1. Effects of Trp to Phe mutation on the fluorescence emission spectra, fluorescence

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quenching and CD. (A) Fluorescence intensities of WT and Trp mutants were recorded upon

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the excitation at 280 nm. (B) Stern-Volmer plots were plotted by recording the maximum fluorescence intensities in the presence of different concentrations of acrylamide, upon

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excitation at 280 nm. F0/F values were plotted against acrylamide concentration. F0, fluorescence intensity in the absence of acrylamide; F, fluorescence intensity in the presence

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of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 M acrylamide. (C) CD spectra of WT and Trp mutants were measured at 25°C. WT (black), W110F (cyan), W159F (green), W208F (red), W236F (orange),

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and W238F (blue).

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Fig. 2. Thermal stability of WT and Trp mutants. Thermal stability was measured at 40°C for 3 min after incubation of WT and Trp mutants at 4, 25, 40, and 50C, every 2 h over a period of 10 h. The data are expressed as a percentage of initial activity remaining after incubation at

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the indicated temperatures. The enzymatic activity at 4°C before incubation is 100%. Experiments were repeated three times. Data correspond to the mean for three experiments.

Fig. 3. CD spectra of WT and W208 mutants. CD spectra were measured with 0.38 µg/ml protein samples in buffer containing 20 mM Tris·HCl, pH 8.0, 25 mM KCl, 0.1 mM EDTA, and 5 % glycerol, by incubating the samples at 4°C (blue) and 40°C (red).

Fig. 4. Acrylamide quenching for WT and W208 mutants. The protein samples (0.38 µg/ml) (in buffer containing 20 mM Tris·HCl, pH 8.0, 25 mM KCl, 0.1 mM EDTA, and 5 % 20

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glycerol) and acrylamide solution were incubated separately at 4C and 30C for 30 min prior to the measurement. Acrylamide was added and the maximum fluorescence intensities were

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recorded upon excitation at 280 nm. To construct the Stern-Volmer plots, F0/F values were plotted against the acrylamide concentration at 4C and 30C, where F0, fluorescence

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intensity in the absence of acrylamide; F, fluorescence intensity in the presence of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 M acrylamide. The plot shows variations of fluorescence quenching between

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4C and 30C obtained by subtracting the regression lines of the Stern-Volmer plots at individual temperatures. WT (black), W208A (cyan), W208F (red), W208K (green) and W208Y

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(orange). Data correspond to the mean  S.D. for three experiments.

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Fig. 5. Structure model of EstK. (A) 3D homology model of EstK. A ribbon representation of

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EstK is shown with 5 Trp residues (green) and catalytic triad (cyan) containing S180, D277, and

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H307. (B) A detailed view of the position of W208 in accordance with catalytic site. (C) A detailed view of hydrogen bond formation between Y208 and D308 which is located on the loop next to catalytic residue H307 in W208Y. (D) Multiple sequence alignments of EstK and its

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homologues. Q3K919 is an esterase from the cold-adapted bacterium Pseudomonas fluorescens Pf0-1. EstE1 and AFEST are hyper-thermophilic esterases. The W208 in EstK and the corresponding residue in homolog esterases are highlighted (grey).

Fig. 6. Thermal stability and temperature-induced unfolding of W208Y. (A) Temperature dependence of the activity of WT and W208 mutants were measured in Buffer B (100 mM Tris·HCl, pH 7.5, 100 mM NaCl, and 0.3% Triton X-100) at the indicated temperatures (0, 10, 20, 30, 40, 50, 60, 70, and 80C) for 3 min with 100 µM of pNPA. Graphs represent the formation of p-nitrophenol per unit enzyme. (B) Temperature-induced unfolding was 21

ACCEPTED MANUSCRIPT measured for W208Y using a spectrofluorometer upon excitation at 280 nm after incubation for 6 min at the indicated temperature. The change in the fluorescence intensities at different

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temperatures was shown as the ratio of fluorescence at 0C (F0) to the fluorescence intensities

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at increasing temperatures (F). Data correspond to the mean  S.D. for three experiments. (C

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and D) Temperature-induced unfolding was measured for WT and W208Y using CD

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spectroscopy upon incubation at the indicated temperature for 6 min.

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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ACCEPTED MANUSCRIPT Highlights

W208 is important for maintaining thermal stability of cold-adapted esterase, EstK.



W208Y showed an increased catalytic site thermal stability.



A hydrogen bond between Y208 and D308 is suggested to stabilize the catalytic site.



Weak catalytic site thermal stability of cold-adapted enzymes could be increased.

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

29