Improving the Thermostability and Catalytic Efficiency of the d-Psicose

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Improving the Thermostability and Catalytic Efficiency of the D-Psicose 3-Epimerase from Clostridium bolteae ATCC BAA-613 Using Site-Directed Mutagenesis Wenli Zhang, Min Jia, Shuhuai Yu, Tao Zhang, Leon Zhou, Bo Jiang, and Wanmeng Mu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01058 • Publication Date (Web): 15 Apr 2016 Downloaded from http://pubs.acs.org on April 20, 2016

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Journal of Agricultural and Food Chemistry

Improving the Thermostability and Catalytic Efficiency of the D-Psicose 3-Epimerase from Clostridium bolteae ATCC BAA-613 Using Site-Directed Mutagenesis

Wenli Zhang †, Min Jia †, Shuhuai Yu †, Tao Zhang

†,‡

, Leon Zhou §, Bo Jiang †,‡,

Wanmeng Mu *,†,‡

†

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.

‡

Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.

§

Roquette America 1003 Commercial St Keokuk, USA 52632

*

To whom correspondence should be addressed. Tel: (86) 510-85919161. Fax: (86)

510-85919161. E-mail: [email protected].

1

ACS Paragon Plus Environment


1

ABSTRACT

2

D-Psicose is a highly valuable rare sugar because of its excellent physiological

3

properties and commercial potential. D-Psicose 3-epimerase (DPEase) is the key

4

enzyme catalyzing the isomerization of D-fructose to D-psicose. However, the poor

5

thermostability and low catalytic efficiency are serious constraints on industrial

6

application. To address these issues, site-directed mutagenesis of Tyr68 and Gly109

7

of the Clostridium bolteae DPEase was performed. Compared with the wild-type

8

enzyme, the Y68I variant displayed the highest substrate-binding affinity and

9

catalytic efficiency, and the G109P variant showed the highest thermostability.

10

Furthermore, the double-site Y68I/G109P variant was generated and exhibited

11

excellent enzyme characteristics. The Km value decreased by 17.9%; the kcat/Km

12

increased by 1.2-fold; the t1/2 increased from 156 min to 260 min; and the melting

13

temperature (Tm) increased by 2.4 °C. Moreover, Co2+ could enhanced the

14

thermostability significantly, including of t1/2 and Tm values. All of these indicated

15

that Y68I/G109P variant would be appropriate for the industrial production of

16

D-psicose.

17 18

KEYWORDS: D-Psicose, D-Psicose 3-epimerase (DPEase), thermostability, catalytic efficiency, site-directed mutagenesis.

19

2


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INTRODUCTION

21

D-Psicose, an epimeric D-fructose at the carbon-3 position, is by definition a rare

22

sugar.1 It is an ultra-low calorie sweetener and poorly absorbed, with 70% of the

23

sweetness and 0.3% of the energy of sucrose.2,3 As a result, this compound is utilized

24

as a functional sugar for diabetics and obese patients.4 Moreover, it also improves the

25

gelling properties of food, increases flavor pleasantness, and reduces oxidation in the

26

Maillard reaction.5-7 Additionally, D-psicose has many advantageous physiological

27

properties, including the ability to reduce the accumulation of intra-abdominal fat,8,9

28

scavenge reactive oxygen species (ROS),10 suppress hepatic lipase activity,11 reduce

29

postprandial glycemic elevation,12,13 improve the metabolism of blood lipids,14

30

protect the nervous system,15 increase insulin resistance,16,17 and treat atherosclerotic

31

diseases.18

32

D-psicose is scarce in nature and difficult to chemically synthesize. Therefore,

33

the likely solution is the bioconversion of D-psicose from D-fructose through ketose

34

3-epimerase. The first member of the ketose 3-epimerases, called D-tagatose

35

3-epimerase (DTEase), was isolated from Pseudomonas cichorii ST-24 with

36

D-tagatose as the optimum substrate. 19,20 To date, several ketose 3-epimerases have

37

been cloned and characterized successively, including the D-psicose 3-epimerase

38

(DPEase) from Agrobacterium tumefaciens,21 the DTEase from Rhodobacter

39

sphaeroides,22 the DPEase from Clostridium cellulolyticum H10,23 the DPEase from

40

Ruminococcus sp. 5_1_39BFAA,24 the L-ribulose 3-epimerase (LREase) from

41

Mesorhizobium loti,25 the DPEase from Clostridium scindens ATCC 35704,26 3



42

DPEase from Clostridium bolteae ATCC BAA-613,27 the DPEase from Clostridium

43

sp. BNL1100,28 and the DPEase from Desmospora sp. 8437.29 Moreover, the crystal

44

structures of P. cichorii DTEase,30 A. tumefaciens DPEase,31 and C. cellulolyticum

45

DPEase32 and M. loti LREase33 have been determined.

46

Generally, high thermostability is required for bioconversion to produce rare

47

sugars because high temperatures increase the utilization efficiency. However, the

48

thermostability of the known DPEases is poor, leading to inefficient bioconversion to

49

produce D-psicose. To date, molecular modification technology has been applied to

50

L-arabinose isomerase and D-glucose isomerase and some desirable variants have

51

already been produced.34,35 However, there were very few similar studies on ketose

52

3-epimerases. In 2011, by the random mutagenesis, the double-site I33L/S213C

53

variant of A. tumefaciens was gained, which displayed excellent improvement in the

54

thermostability.36 Recently, Bosshart et al. reported the directed divergent evolution

55

of P. cichorii DTEase. By iterative randomization and screening around the

56

substrate-binding site, the eight-site mutant IDF8 was achieved, which showed

57

9-fold improved kcat for D-fructose 37

58

In previous study, the wild-type C. bolteae DPEase was cloned and characterized;

59

this enzyme can be distinguished from other DPEases by its optimal neutral pH (pH

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7.0).27 However, it was not stable above 50 °C, which limited its practical

61

application. In the present study, the site-directed mutagenesis technique was applied

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to improve the thermostability and catalytic efficiency of the wild-type C. bolteae

63

DPEase. The enzymatic properties of the variant enzymes were determined and 4


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compared with the wild-type enzyme. Moreover, the structure-function relationships

65

were explored based on homology modeling.

66 67

MATERIALS AND METHODS

68

Bacterial Strains, Plasmids, and Enzymes. Escherichia coli DH5α was used as

69

the host for cloning, and E. coli BL21(DE3) was used as the host for the

70

overexpression of DPEase. Both of the strains were purchased from Sangon

71

Biological Engineering Technology and Services (Shanghai, China). The pET-22b(+)

72

plasmid, obtained from Novagen (Darmstadt, Germany), was used for sub-cloning.

73

The DPEase gene of Clostridium bolteae ATCC BAA-613 (GenBank accession

74

number EDP19602) was synthesized by Generay Biotechnology Co., Ltd. (Shanghai,

75

China) based on the preferred codon usage of E. coli.

76 77

Site-directed Mutagenesis. Site-directed mutagenesis of the DPEase gene of C.

78

bolteae was accomplished using a one-step PCR method using a pair of synthetic

79

complementary oligonucleotides as primers (Table S1). The recombinant plasmid

80

pET-CB-dpe containing the wild-type DPEase gene of C. bolteae was used as the

81

template. The amplified PCR products were digested using DpnI and cloned into the

82

plasmid, which was used to transform E. coli DH5α host cells. The sequences of the

83

constructed variant genes were verified by DNA sequencing. To obtain expression

84

strains, plasmids containing the correct variant genes were reintroduced into E. coli

85

BL21(DE3) cells. 5



86 87

Heterologous Expression and Purification of the Enzymes. For heterologous

88

expression of the wild-type and variant enzymes, the E. coli BL21(DE3) cells that

89

had been transformed with the target genes were cultured aerobically in

90

Luria-Bertani (LB) medium supplemented with 100 µg/mL ampicillin at 37 °C, with

91

vigorous agitation (200 rpm), until the A600 reached 0.5 to 0.6. Then, the recombinant

92

strains were induced at 28 °C using isopropyl β-D-1-thiogalactopyranoside (IPTG) at

93

a final concentration of 1 mM for an additional 6 h. The induced cells were collected

94

from the culture medium by centrifugation at 12,000×g for 15 min at 4 °C and were

95

stored at -20 °C.

96

To purify the recombinant wild-type and variant enzymes, the pelleted cells were

97

re-suspended in 50 mM sodium phosphate buffer (pH 7.0) and then were disrupted

98

by sonication for 12 min (pulse on for 2 s and pulse off for 3 s). The lysates were

99

centrifuged (12,000 ×g, 30 min, 4 °C) to remove the cellular debris. After that, the

100

cleared supernatants were filtered through a 0.22-µm filter, and the each sample was

101

loaded onto a column containing Ni2+-chelating Sepharose Fast Flow resin (Uppsala,

102

Sweden) that had been pre-equilibrated using buffer A (50 mM sodium phosphate

103

buffer, 500 mM NaCl, pH 7.0). Buffer B (50 mM sodium phosphate buffer, 500 mM

104

NaCl, 50 mM imidazole, pH 7.0) was applied to elute proteins that were

105

nonspecifically bound to the resin and finally, buffer C (50 mM sodium phosphate

106

buffer, 500 mM NaCl, 500 mM imidazole, pH 7.0) was used to elute the bound

107

target protein from each column. The fractions exhibiting DPEase activity were 6


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pooled and were dialyzed overnight against 50 mM sodium phosphate buffer (pH 7.0)

109

containing 10 mM ethylenediamine tetraacetic acid (EDTA) to remove the metal

110

ions. Immediately afterward, each protein was dialyzed against 50 mM EDTA-free

111

sodium phosphate buffer (pH 7.0) for 24 h to remove the EDTA. All of the

112

purification steps were performed at 4 °C.

113

The protein concentrations we determined using the standard Lowry method,

114

using bovine serum albumin as the standard. The purity and molecular mass of the

115

recombinant

116

sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue

117

R-250 staining.

DPEases

were

determined

using

12%

sodium

dodecyl

118 119

Enzymatic Activity and Kinetic Parameter Assays. The activity of the

120

wild-type enzyme and the variant enzymes were routinely assayed by determining

121

the amount of D-psicose that had been converted from D-fructose using

122

high-performance liquid chromatography (HPLC). The activities of the wild-type

123

and variant enzymes were determined in 50 mM sodium phosphate buffer (pH 7.0)

124

containing 0.4 mM Co2+, using 50 g/L D-fructose as the substrate, at 55 °C for 5 min,

125

and the samples were immediately heated in a boiling water bath for 10 min to

126

inactivate the enzymes. Before being subjected to HPLC, the samples were

127

centrifuged at 10000 ×g for 30 min, diluted 1:4 with deionized water, and then

128

filtered through a 0.22-µm filter. The concentration of the enzymes in the reaction

129

system was 0.5 µM. 7



130

The values for the kinetic parameters, including the Michaelis-Menten constant

131

(Km), the turnover number (kcat), and the catalytic efficiency (kcat/Km) were measured

132

by using Lineweaver-Burk equation, with D-fructose and D-tagatose as the

133

substrates (5 to 400 mM) at 55 °C in 50 mM sodium phosphate buffer (pH 7.0)

134

containing 0.4 mM Co2+ and 0.5 µM enzyme. All of reported values of the kinetic

135

parameters are the averages derived from triplicate measurements.

136 137

Effect of pH on the Enzymatic Activity. To evaluate the effect of pH on the

138

epimerization reaction, the DPEase activities were determined using D-fructose as

139

the substrate, as described above, across a pH range of 5.0 to 9.0, using 50 mM

140

sodium acetate buffer (pH 5.0 to 5.5), 50 mM sodium phosphate buffer (pH 6.0 to

141

7.0), and 50 mM Tris-HCl buffer (pH 7.5 to 9.0). For the pH stability tests, the

142

enzymes were pre-incubated in buffers with the various pH values (5.0 to 9.0) at

143

4 °C for 2 h. The residual activities of each enzyme were determined under the

144

standard assay conditions at 55 °C and were plotted as a percentage of the initial

145

activity (100%).

146 147

Effect of Temperature on the Enzymatic Activity. To investigate the optimum

148

reaction temperature, the activity of the DPEases in 50 mM sodium phosphate buffer

149

at pH 7.0 at 40 to 80 °C was determined. At each temperature, the enzymatic

150

reaction was initiated by adding recombinant enzyme to the pre-incubated mixture to

151

achieve a final concentration of 0.5 µM. 8


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To assess their thermostability, the half-life (t1/2) at 55 °C and melting temperature

153

(Tm) valuses were determined. For t1/2 determination, the enzymes were diluted using

154

50 mM sodium phosphate buffer (pH 7.0) to a concentration of 10 µM and then were

155

pre-incubated at 55 °C with and without 1 mM Co2+. At given times, samples were

156

withdrawn and the residual activity was determined at 55 °C in the same buffer. The

157

initial activity level before pre-incubation at 55 °C was defined as 100%.

158

For Tm determination, differential scanning calorimetry (DSC) measurements were

159

performed using a high sensitivity Nano-DSC (TA Instruments, New Castle, USA).

160

Before being loaded into the DSC cell, the enzymes and buffers were freshly

161

prepared, and extensively degassed for 10 min under vacuum. The reference and

162

sample cells were then filled with sodium phosphate buffer PBS buffer (50 Mm, Ph

163

6.0) and the enzyme solutions, respectively. After an equilibration period of 300 s,

164

the cells were heated from 25 °C to 100 °C at 3 atm with a temperature ramp of 1 °C

165

/min. DSC data were analyzed using TA Instruments NanoAnalyze software, and the

166

observed thermograms were baseline-corrected.

167 168

Bioconversion of D-fructose to D-psicose. The conversion of D-fructose to

169

D-psicose was performed with purified enzyme (0.5 µM) at the optimal reaction

170

conditions (pH 7.0, 55 °C) with the addition of 1 mM Co2+. The initial concentration

171

of 500 g/L D-fructose was used for D-psicose bioconversion with 1 liter reaction

172

system.

173 9



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Homology Modeling and Structure Energy Minimization. To achieve an

176

appropriate template, the amino acid sequence of the wild-type C. bolteae DPEase

177

was submitted to the SWISS-MODEL protein-modeling server, using Automated

178

Mode method (http://www.expasy.ch/swissmod/SWISS-MODEL.html).38,39 Then,

179

the X-ray crystal structure of C. cellulolyticum DPEase (PDB ID, 3vnk) bound to

180

D-fructose was selected as the template, and the theoretical three-dimensional

181

homology models of both the wild-type and variant enzymes were created. The

182

Ramachandran map created by WinCoot was used to evaluate the stereochemical

183

quality of homology models; and the compatibility of an atomic model (3D) with the

184

amino

185

(http://services.mbi.ucla.edu/SAVES/).40

acid

sequence

(1D)

was

analyzed

by

VERIFY-3D

186

The structure energy minimization was performed using the Discovery Studio

187

software. The bound substrate D-fructose was obtained from the PDB file (PDB:

188

3VNK) of C. cellulolyticum DPEase and D-fructose complexs. Then, the bound

189

substrate can be introduced into the homology modeling structure of C. bolteae

190

DPEase

191

by the structure energy minimization calculation. The CHARMm force field was

192

applied and the minimization cycles were 200. Finally, the molecular models were

193

visualized by utilizing the PyMol software.

by superimposing the X-ray structure of the D-fructose complex, followed

194 195

RESULTS AND DISCUSSION 10


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Alanine-scanning Mutagenesis. Up to date, several DPEases have been cloned

197

and characterized. So, the amino acid sequences alignment of DPEases was carried

198

out (Fig. 1). As previously speculated, Glu152 and Glu246 are involved in

199

deprotonating and protonating at the C-3 epimerization; Glu158, His188 and Arg217

200

were involved in the binding of the D-fructose through hydrogen bonding; Tyr68 and

201

Gly109 were involved in the recognition of the specific substrate. 31-33,41,42 Based on

202

the results of the multiple sequences alignment and the previous investigation, the

203

potential key residues in the active site of C. bolteae DPEase were predicted and

204

were selected for mutagenesis, including Tyr68, Gly109, Glu152, Glu158, His188,

205

Arg217 and Glu246. Because of its small size, simple structure, and lack a functional

206

group, alanine (Ala) was used to substitute the key residues. The E152A, E158A,

207

H188A, R217Aand E246A variant enzymes displayed no detectable catalytic activity,

208

whereas the Y68A and G109A variant enzymes exhibited 20% and 68% of the

209

activity of the wild-type enzyme, respectively (data not shown). The probable cause

210

for these results was that when the Glu152, Glu158, His188, Arg217 and Glu246

211

amino-acid residues were substituted by Ala, the level of steric hindrance was

212

reduced and the distances between the substrate were expanded, hampering

213

hydrogen-bond formation. Analysis of the sequence alignment revealed that Glu152,

214

Glu 158, His 188, Arg 217 and Glu 246 were the key residues in the active site and

215

they were strictly conserved in members of the ketose 3-epimerase family, whereas

216

Tyr68 and Gly109 were not conserved residues. Thus, Tyr68 and Gly109 were

217

selected for site-directed mutagenesis to obtain variant enzymes with the desired 11



218

properties.

219 220

Site-directed Mutagenesis of Tyr68 and Gly109. From the analysis of the

221

sequence alignment and the results of Ala-scanning mutagenesis, it could be

222

concluded that although Tyr68 and Gly109 affected the catalytic activity, they did

223

not play key roles in this property. Hence, it was predicted that Tyr68 and Gly109

224

were involved in the recognition of the specific substrate and that mutating these

225

residues might change the shape of the hydrophobic pocket of the enzyme. A similar

226

situation existed for the L-arabinose isomerase of Bacillus stearothermophilus (B.

227

stearothermophilus L-AI) and Bacillus licheniformis (B. licheniformis L-AI), which

228

had different active residues and differently shaped active-site pockets. The results of

229

studies of these enzymes revealed the basis of their substrate specificity; for example,

230

the B. stearothermophilus L-AI enzyme specifically catalyzed the isomerization of

231

D-galactose and L-arabinose, whereas the B. stearothermophilus L-AI enzyme could

232

catalyze only the isomerization of L-arabinose. 43,44

233

Tyr68 and Gly109 were selected for site-directed mutagenesis to obtain variant

234

enzymes with a higher affinity for D-fructose. Tyr68 and Gly109 were substituted by

235

other hydrophobic amino acids, including valine (Val), phenylalanine (Phe), proline

236

(Pro), isoleucine (Ile) and leucine (Leu) and Y68V, Y68F, Y68P, Y68I, Y68L, G109V,

237

G109F, G109P G109L and G109L variant constructs were created. The variant

238

enzymes were purified and their enzymatic activities using D-fructose or D-tagatose

239

as substrates were determined. 12


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As shown in Table 1, most of the variant enzymes showed activity using

241

D-fructose as the substrate, except for the G109F variant, but when D-tagatose was

242

used as the substrate, the activity of all of variant enzymes was significantly reduced,

243

sometimes to an undetectable level. The results suggested that the pocket shape was

244

changed by the changes in the residues, which affected the substrate specificity.

245

To obtain enzymes with a relatively greater level of activity, Y68I, Y68F, G109A,

246

G109P and Y68I/G109P double-site mutation constructs were purified and assayed

247

by SDS-PAGE (Fig. S1). Furthermore, enzymatic activity and kinetic analytic assays

248

of the resultant enzymes were conducted.

249 250

Characterization of the Wild-type Enzyme and the Variant Enzymes. In the

251

present study, the effect of pH on the epimerization by the wild-type enzyme and the

252

variant enzymes was determined across the pH range of 5.0 to 9.0 at 55 °C. The

253

experimental results showed that the optimum pH of the wild-type and the Y68I,

254

Y68F, G109A, G109P and Y68I/G109P variant enzymes were the same (pH 7.0).

255

However, the G109A variant exhibited more than 90% of its maximal activity at pH

256

6.0, which was higher than the activity of the other enzymes at this pH (Fig. 2A).

257

Compared with the wild-type enzyme, the five variant enzymes displayed no

258

significant difference in the pH stability tests when they were pre-incubated in

259

solutions with various pH values (5.0 to 9.0) at 4 °C for 2 h (Fig. 2B).

260

The optimal reaction temperatures of the wild-type enzyme and the variant

261

enzymes in the temperature range of 40 to 80 °C in 50 mM sodium phosphate buffer 13



262

(pH 7.0) were also investigated, using D-fructose as the substrate. The results

263

showed that the optimal temperature of the Y68F and G109A variant enzymes was

264

55 °C, which was the same as that of the wild-type enzyme, whereas the optimal

265

temperature of the Y68I, G109P and Y68I/G109P variant enzymes was increased

266

from 55 to 60 °C (Fig. 3A). At 60 °C, the wild-type enzyme displayed 80% of its

267

maximal enzymatic activity, while the Y68F and G109A variants exhibited 82.9%

268

and 85.1% of their maximum activity, respectively. Furthermore, at 80 °C, wild-type

269

enzyme displayed only 32.6% of its maximum activity, while the Y68I, G109P and

270

Y68I/G109P variants displayed 33.0%, 58.1%, and 83.1% of their individual

271

maximum activities, respectively.

272

To assess the thermostability of the wild-type and variant enzymes, their t1/2 and

273

Tm values were determined, respectively. For t1/2 measurement, the enzymes were

274

pre-incubated at 55 °C in 50 mM sodium phosphate buffer (pH 7.0) with and without

275

1 mM Co2+. After incubation for different periods, samples were withdrawn and the

276

residual activity was determined. As shown in Table 2, upon adding Co2+ to a final

277

concentration of 1 mM during 55 °C incubation, both the wild-type enzyme and the

278

variant enzymes displayed higher thermostability levels. When incubated in the

279

presence of Co2+, the t1/2 value of the wild-type enzyme was 156 min. In comparison,

280

the Y68I, Y68F, and G109A variant enzymes displayed shorter t1/2 value, which were

281

122.8 min, 73.2 min, and 112.1 min, respectively. However, the G109P and

282

Y68I/G109P variant enzymes were more thermostable and the t1/2 value of the

283

G109P variant was 327.4 min and that of the Y68I/G109P variant was 264 min, 14


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284

which were 2.1- and 1.7-fold that of the wild-type enzyme, respectively.

285

Subsequently, the melting temperature (Tm) of the wild-type and variant

286

enzymes was determined using Nano-DSC (Fig. S2). The thermogram given in Fig.

287

3B showed representative examples of DSC data (wild-type and Y68I/G109P variant

288

enzymes). The single endothermic transition peak was observed by Nano-DSC,

289

which suggested a two-state mechanism of unfolding (Fig. 3B).45 As shown in Table

290

2, the Tm value for wild-type enzyme was measured to be 54.6 °C. The Y68I, Y68F,

291

and G109A variant enzymes possessed a slightly lower thermostability with Tm

292

values of 53.5 °C, 51.1 °C and 51.7 °C, respectively; while the G109P and

293

Y68I/G109P variant enzymes exhibited enhanced thermostability with Tm values of

294

59.3 °C and 55.4 °C, respectively. Additionally, it clearly appeared that the presence

295

of Co2+ (1 mM) displaces the Tm in an upward direction; the least shift, ∆Tm, was

296

6.4 °C (Table 2). After adding Co2+, the increasing trends of Tm values were

297

analogous to the changes of t1/2 values. In general, the rise of Tm values further

298

confirmed that Co2+ could enhance the thermostability of C. bolteae DPEase. Similar

299

result existed in C. scindens DPEase, that Mn2+ could improve the structural stability

300

during both heat- and urea-induced unfolding.26

301 302

Enzyme Kinetics. The values for the kinetic parameters of the wild-type enzyme

303

and the variant enzymes at 55 °C were determined using Lineweaver-Burk equation,

304

and the results are shown in Table 3. Using D-fructose as the substrate, the Km value

305

of the Y68I and Y68I/G109P variant enzymes was decreased by 23.0% and 17.9% 15



306

compared with that the wild-type enzyme, and the catalytic efficiency (kcat/Km)

307

values were increased to 1.4- and 1.2-fold that of wild-type enzyme, respectively. In

308

contrast, the Km values of the Y68F, G109A and G109P variant enzymes were

309

increased by 20.9%, 14.0% and 44.0%, respectively, and the kcat/Km values were

310

reduced to 68.6%, 63.0% and 52.2%, respectively. When D-tagatose was used as the

311

substrate, the Km values of all of the variant enzymes were increased substantially,

312

by 3.6-fold for the Y68I variant, 4.9-fold for the Y68F variant, 2.5-fold for the

313

G109A variant, 2.0-fold for the G109P variant, and 2.8-fold for the Y68I/G109P

314

variant. In addition, the kcat/Km values of all of the variant enzymes were decreased

315

to different degrees. Using D-tagatose as the substrate, the relative activity of all

316

variants sharply reduced or even lost (Tbale 1). Compared with that the wild-type

317

enzyme, the Km values of variants obviously increased, and he kcat/Km values were

318

significantly reduced (Tbale 2).

319 320

Bioconversion of D-fructose to D-psicose. The high-level production of

321

D-psicose from D-fructose was shown in Fig. 4. For the wild-type enzyme and

322

Y68I/G109P variant, 93 and 137 g/L D-psicose was produced from 500 g/L

323

D-fructose, respectively. In addition, the reaction speed of Y68I/G109P variant was

324

also higher than the wild-type enzyme. From the results, it further verified that,

325

Y68I/G109P variant had higher thermostability and catalytic efficiency than

326

wild-type enzyme. Y68I/G109P variant would be appropriate for the industrial

327

production of D-psicose. 16


Page 16 of 40

Page 17 of 40


328 329

Homology Building. To date, 4 crystal structures of DPEase have been solved,

330

and their catalytic mechanisms have been preliminarily explored, including the P.

331

cichorii DTEase,30 A. tumefaciens DPEase,31 C. cellulolyticum DPEase32 and M. loti

332

LREase33. Homology molecular modeling of C. bolteae DPEase (as shown in Fig 5A)

333

and Y68I/G109P variant were conducted using the crystal structure of C.

334

cellulolyticum DPEase (PDB ID, 3vnk) as the template, which showed the highest

335

identity (51.6%). Then the conducted model was evaluated by Ramachandran plot

336

(Fig. S3). As shown in Fig. S3, there were 93.01% of amino acid residues located in

337

preferred regions, 5.24% located in allowed regions, and 1.75% located in outlier

338

regions. VERIFY-3D analysis showed that 92.01% (larger than 80%) of the amino

339

acids had an average score ≥ 0.2 in 3D/1D profile. All these results showed that

340

the predicted models were suitable. The overall structures of C. bolteae DPEase and

341

its variants were extremely similar to the crystal structure of C. cellulolyticum

342

DPEase.32 The enzyme was tetramer and each subunit displayed a (β/α)8 TIM barrel.

343

As displayed in Fig 5B, the monomer of C. bolteae DPEase model (green)

344

superimposed with the vast majority regions of C. cellulolyticum DPEase (pink). The

345

criteria for selecting the active-site residues were based on the analysis of solved

346

crystal structure and amino acid sequence alignment. As shown in Fig. 5C, the active

347

site residues of both C. bolteae DPEase and C. cellulolyticum DPEase were very

348

similar. The tiny differences appeared in the residues at Tyr68 and Gly109 positions

349

in C. bolteae DPEase; while the residues at the similar positions of C. cellulolyticum 17



350

DPEase were Gly65 and Ala107. It indicated that the active sites of both C. bolteae

351

and C. cellulolyticum DPEase were similar.

352 353

Structure Energy Minimization. The structure energy minimization was

354

completed by Discovery Studio, and the results were visualized by PyMol software.

355

Structure modeling revealed that the residue in position 68 was located in linkage of

356

the β3-α3 loop in the active-site pocket, which affected the shape of this

357

hydrophobic pocket. The residue at the 109 position was also located in a loop

358

region of the β-4 turn of the active site, which has been recognized as a significantly

359

disordered region using RONN software. When Tyr68 and Gly109 were substituted

360

by Ile68 and Pro109, the distance between active site and bound D-fructose became

361

closer. Moreover, a new hydrogen bond between residue at 68 positions and O6 of

362

D-fructose were formed. These effects led to the higher thermostability and catalytic

363

efficiency for Y68I/G109P variant.

364

The Tyr68 of wild-type enzyme could not form hydrogen bonds with D-fructose,

365

while Y68I/G109P variant could form a new hydrogen bond with O6 of D-fructose

366

(Fig. 6). In addition, the distance between D-fructose and active site residues became

367

much closer, indicating that binding interaction between Y68I/G109P variant and

368

D-fructose was much stronger than that of wild-type enzyme. At the same time, the

369

side chain of Ile66 had hydrophobic interactions with Pro70, which was consistent

370

with the results of Kim et al.32,41 The hydrophobic active-site pocket that formed in

371

this variant enzyme made it easier to bind to D-fructose and increased the substrate 18


Page 18 of 40

Page 19 of 40


372

binding affinity. Compared with D-tagatose, D-fructose bonded more closely to the

373

active site. The changes in the shape of the active site caused by substituting the Y68

374

residue might hinder the binding of the enzyme to D-tagatose (Table 1). Interestingly,

375

the affinities of the Y68I and Y68F variant enzymes for D-fructose were similar to

376

that of the wild-type enzyme, whereas the affinities of the variant enzymes for

377

D-tagatose were dramatically decreased. It was predicted that when binding with

378

D-fructose, the surface area of the hydrophobic pocket between O6 and the residue

379

in position 66 was slightly changed, but when binding with D-tagatose, the surface

380

area of this region was decreased. The substrate-binding affinity of the enzyme could

381

thus be adjusted according to the conformational flexibility of the loop region.

382

The residue in the 109 position of the wild-type enzyme is Gly (Fig. 6A), which

383

lacks a side-chain group. Thus, having Gly in the 109 position rather than another

384

amino acid would increase the conformational flexibility of the enzyme. Variant

385

enzymes with hydrophobic substitutions of Pro at the 109 position were created (Fig.

386

6B) and the results of the assays showed that their level of thermostability was

387

significantly increased. Pro contains a pyrrolidine side-chain, which tends to reduce

388

the flexibility of DPEase, and increase the structure stability of hydrophobic active

389

sites. Thus the thermostability of Y68I/G109P variant was significantly enhanced. 32

390 391

ASSOCIATED CONTENT

392

Supporting Information

393

Fig. S1 SDS-PAGE of C. bolteae DPEase and its variants. Lane M, protein marker; 19



394

lane 1, Wild-type enzyme; lane 2, Y68I variant; lane 3, Y68F variant; lane 4, G109A

395

variant; lane 5, G109P variant; lane 6, Y68I/ G109P variant. Fig. S2 Thermal

396

unfolding of wild-type and variant enzymes monitored by Nano-DSC. The curves in

397

black and red colors represented the enzymes without or with 1 mM Co2+,

398

respectively. Fig. S3 Ramachandran plot of the C. bolteae DPEase model analyzed

399

by WinCoot. Table S1 Primers of site-specific mutagenesis. This material is

400

available free of charge via the Internet at http://pubs.acs.org.

401 402

ACKNOWLEDGMENTS

403

Corresponding Author

404

* E-mail: [email protected]. Phone: (86) 510-85919161. Fax: (86)

405

510-85919161.

406 407

Fundings

408

This study was supported by grants from the NSFC Project (No. 31171705 and

409

21276001), the 863 Project (No. 2013AA102102), the Fundamental Research Funds

410

for the Central Universities (No. JUSRP51304A), and the Support Project of Jiangsu

411

Province (No. BK20130001).

412

Notes

413

The authors declare that they have no conflict of interest.

414 415

ABBREVIATIONS USED 20


Page 20 of 40

Page 21 of 40


416

DPEase, D-psicose 3-epimerase; t1/2, half-life; Tm, melting temperature; DTEase,

417

D-tagatose 3-epimerase; L-ribulose 3-epimerase, LREase; LB, Luria-Bertani; IPTG,

418

isopropyl β-D-1-thiogalactopyranoside; EDTA, ethylenediamine tetraacetic acid;

419

SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC,

420

high-performance liquid chromatography; DSC, differential scanning calorimetry.

421

21



422

Page 22 of 40

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457

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(15) Takata, M. K.; Yamaguchi, F.; Nakanose, Y.; Watanabe, Y.; Hatano, N.;

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Tsukamoto, I.; Nagata, M.; Izumori, K.; Tokuda, M. Neuroprotective effect of

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Matsunaga, T.; Tsukamoto, I.; Yamaguchi, F.; Kamitori, K.; Dong, Y. Y.; Hirata, Y.;

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Murao, K.; Toyoda, Y.; Tokuda, M. Rare sugar D-psicose improves insulin

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Fatty (OLETF) rats. Biochem. Bioph. Res. Commun. 2011, 405, 7-12.

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Y.; Sui, L.; Tsukamoto, I.; Ueno, M.; Tokuda, M. Rare sugar D-psicose protects

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pancreas beta-islets and thus improves insulin resistance in OLETF rats. Biochem.

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N.; Li, J.; Ahmed, R. A. M.; Matsumoto, K.; Nishiuchi, T.; Tokuda, M.; Ishida, T.

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D-Psicose inhibits the expression of MCP-1 induced by high-glucose stimulation in

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HUVECs. Life Sci. 2007, 81, 592-599.

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(19) Izumori, K.; Khan, A. R.; Okaya, H.; Tsumura, T. A new enzyme,

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D-ketohexose 3-epimerase, from Pseudomonas sp. ST-24. Biosci. Biotechnol. Bioch.

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1993, 57, 1037-1039. 24


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(20) Itoh, H.; Okaya, H.; Khan, A. R.; Tajima, S.; Hayakawa, S.; Izumori, K.

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Purification and characterization of D-tagatose 3-epimerase from Pseudomonas sp.

490

ST-24. Biosci. Biotechnol. Bioch. 1994, 58, 2168-2171.

491

(21) Kim, H. J.; Hyun, E. K.; Kim, Y. S.; Lee, Y. J.; Oh, D. K. Characterization of

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an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to

493

D-psicose. Appl. Environ. Microbiol. 2006, 72, 981-985.

494

(22) Zhang, L. T.; Mu, W. M.; Jiang, B.; Zhang, T. Characterization of

495

D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into

496

D-psicose. Biotechnol. Lett. 2009, 31, 857-862.

497

(23) Mu, W. M.; Chu, F. F.; Xing, Q. C.; Yu, S. H.; Zhou, L.; Jiang, B. Cloning,

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expression, and characterization of a D-psicose 3-epimerase from Clostridium

499

cellulolyticum H10. J. Agric. Food Chem. 2011, 59, 7785-7792.

500

(24) Zhu, Y. M.; Men, Y.; Bai, W.; Li, X. B.; Zhang, L. L.; Sun, Y. X.; Ma, Y. H.

501

Overexpression of D-psicose 3-epimerase from Ruminococcus sp. in Escherichia

502

coli and its potential application in D-psicose production. Biotechnol. Lett. 2012, 34,

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1901-1906.

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(25) Uechi, K.; Takata, G.; Fukai, Y.; Yoshihara, A.; Morimoto, K. Gene cloning

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and characterization of L-ribulose 3-epimerase from Mesorhizobium loti and its

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application to rare sugar production. Biosci. Biotechnol. Bioch. 2013, 77, 511-515.

507

(26) Zhang, W. L.; Fang, D.; Xing, Q. C.; Zhou, L.; Jiang, B.; Mu, W. M.

508

Characterization of a novel metal-dependent D-psicose 3-epimerase from

509

Clostridium scindens 35704. PLoS ONE 2013, 8. 25



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(27) Jia, M.; Mu, W. M.; Chu, F. F.; Zhang, X. M.; Jiang, B.; Zhou, L. L.; Zhang, T.

511

A D-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for

512

D-psicose production: cloning, expression, purification, and characterization. Appl.

513

Microbiol. Biotechnol. 2014, 98, 717-725.

514

(28) Mu, W. M.; Zhang, W. L.; Fang, D.; Zhou, L.; Jiang, B.; Zhang, T.

515

Characterization of a D-psicose-producing enzyme, D-psicose 3-epimerase, from

516

Clostridium sp. Biotechnol. Lett. 2013, 35, 1481-1486.

517

(29) Zhang, W. L.; Fang, D.; Zhang, T.; Zhou, L.; Jiang, B.; Mu, W. M.

518

Characterization of a metal-dependent D-psicose 3-Epimerase from a Novel Strain,

519

Desmospora sp. 8437. J. Agric. Food Chem. 2013, 61, 11468-11476.

520

(30) Yoshida, H.; Yamada, M.; Nishitani, T.; Takada, G.; Izumori, K.; Kamitori, S.

521

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its

522

complexes with D-tagatose and D-fructose. J. Mol. Biol. 2007, 374, 443-453.

523

(31) Kim, K.; Kim, H. J.; Oh, D. K.; Cha, S. S.; Rhee, S. Crystal structure of

524

D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true

525

substrate D-fructose: a pivotal role of metal in catalysis, an active site for the

526

non-phosphorylated substrate, and its conformational changes. J. Mol. Biol. 2006,

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361, 920-931.

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(32) Chan, H. C.; Zhu, Y. M.; Hu, Y. M.; Ko, T. P.; Huang, C. H.; Ren, F. F.; Chen,

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C. C.; Ma, Y. H.; Guo, R. T.; Sun, Y. X. Crystal structures of D-psicose 3-epimerase

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from Clostridium cellulolyticum H10 and its complex with ketohexose sugars.

531

Protein Cell 2012, 3, 123-131. 26


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(33) Uechi, K.; Sakuraba, H.; Yoshihara, A.; Morimoto, K.; Takata, G. Structural

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insight into L-ribulose 3-epimerase from Mesorhizobium loti. Acta Crystallogr. D.

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2013, 69, 2330-2339.

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(34) Lee, S.J.; Lee, S. J.; Lee, Y.J.; Kim, S.B.; Kim, S.K.; Lee, D.W. Homologous

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alkalophilic and acidophilic

L-arabinose isomerases reveal region-specific

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contributions to the pH dependence of activity and stability. Appl. Environ.

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Microbiol. 2012, 78, 8813-8816.

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(35) Hartley, B. S.; Hanlon, N.; Jackson, R. J.; Rangarajan, M. Glucose isomerase:

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insights into protein engineering for increased thermostability. Biochim. Biophy. Acta

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Protein Struct. Mol. Enzym. 2000, 1543, 294-335.

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(36) Choi, J.G.; Ju, Y.H.; Yeom, S.J.; Oh, D.K. Improvement in the thermostability

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of D-psicose 3-epimerase from Agrobacterium tumefaciens by random and

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site-directed mutagenesis. Appl. Environ. Microbiol. 2011, 77, 7316-7320.

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(37) Bosshart, A.; Hee, C. S.; Bechtold, M.; Schirmer, T.; Panke, S. Directed

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divergent evolution of a thermostable D-tagatose epimerase towards improved

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activity for two hexose substrates. Chembiochem. 2015, 16, 592-601.

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(38) Guex, N.; Peitsch, M.C.; Schwede, T. Automated comparative protein structure

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modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective.

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Electrophoresis. 2009, 30, S162-S173.

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(39) Biasini, M.; Bienert, S.; Waterhouse, A. Arnold, K. Studer, G.; Schmidt, T.;

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Kiefer, F.; Cassarino, T. G.; Bertoni, M.; Bordoli, L.; Schwede, T. SWISS-MODEL:

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modelling protein tertiary and quaternary structure using evolutionary information. 27



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(41) Kim, H. J.; Lim, B. C.; Yeom, S. J.; Kim, Y. S.; Kim, D.; Oh, D. K. Roles of

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Ile66 and Ala107 of D-psicose 3-epimerase from Agrobacterium tumefaciens in

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binding O6 of its substrate, D-fructose. Biotechnol. Lett. 2010, 32, 113-118.

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(42) Kim, H. J.; Yeom, S. J.; Kim, K.; Rhee, S.; Kim, D.; Oh, D. K. Mutational

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analysis of the active site residues of a D-psicose 3-epimerase from Agrobacterium

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tumefaciens. Biotechnol. Lett. 2010, 32, 261-268.

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569 570

(45) Daggett, V.; Fersht, A. The present view of the mechanism of protein folding. Nat. Rev. Mol. Cell Biol. 2003, 4, 497-502.

28


Page 28 of 40

Page 29 of 40


Figure legends Fig. 1 Multiple sequence alignment of DPEases from various microorganisms. The origins of DPEase enzymes with the GenBank accession numbers as follows: Clce-DPEase (C. cellulolyticum DPEase, ACL75304), Clbo-DPEase (C. bolteae DPEase, EDP19602), Agtu-DPEase (A. tumefaciens DPEase, AAK88700.1), Clsc-DPEase (C. scindens DPEase, EDS06411.1), Clsp-DPEase (Clostridium sp. DPEase,

YP_005149214.1),

Desp-DPEase

(Desmospora

sp.

DPEase,

WP_009711885.1), and Rusp-DPEase (Ruminococcus sp. DPEase, ZP04858451).

Fig. 2 Effects of different pH values on the activities and stabilities of the wild-type C. bolteae DPEaseand its variants. (A) Optimum pH of the wild-type enzyme and the variant enzymes. The enzymatic activities were determined at 55 °C for 5 min in buffers with different pH (5.0 to 9.0) values, in the presence of 0.4 mM Co2+. (B) The pH stability of the enzymes was tested by pre-incubating the purified enzymes in solutions with various pH values (pH 5.0 to 9.0) at 4 °C for 2 hours. All of the assays were performed in triplicate.

Fig. 3 Effects of different temperatures on the activity and thermostability of the wild-type C. bolteae DPEase and Y68I/G109P variants. (A) Optimum temperature of the wild-type and variant enzymes. The enzyme activities were determined in 50 mM sodium phosphate buffer (pH 7.0) at between 40 and 80 °C for 5 min, in the

29



presence of 0.4 mM Co2+. All of the assays were performed in triplicate. (B) The DSC datas of wild-type and Y68I/G109P variant enzymes with 1mM Co2+.

Fig. 4 D-Psicose production from D-fructose by wild-type and Y68I/G109P enzymes. All the reactions were performed at pH 7.0 and 55 °C, containing 0.5 µM enzyme,1 mM Co2+ and 500 g/L of D-fructose. Results are the mean values of three experiments.

Fig. 5 Three-dimensional model of predicted by SWISS-MODEL. (A) A model structure of C. bolteae DPEase using the crystal structure of C. cellulolyticum DPEase as template. (B) Superimposition of the monomer of C. bolteae DPEase (green) and C. cellulolyticum DPEase (pink). (C) The superposition of active site from C. bolteae DPEase (yellow) and C. cellulolyticum DPEase (cyan).

Fig. 6 Putative active site of the wild-type C. bolteae DPEase (A) and the double-site Y68I/G109P variant DPEase (B). The hydrogen bonds of amino residues at 68 position were shown using green dotted lines.

30


Page 30 of 40

Page 31 of 40


Tables Table 1. Relative specific activity for D-fructose and D-tagatose of the wild-type and the variant enzymes with substitutions at positions 68 and 109. Enzyme

Relative activity (%) D-Fructose (%)

D-Tagatose (%)

Wlid-type

100±2.7

100±2.7

Y68A

19.9±2.2

9.3±1.3

Y68L

9.1±0.71

ND a

Y68I

153.8±6.8

12.4±1.7

Y68P

43.4±3.2

24.6±2.6

Y68F

76.9±1.4

4.3±0.74

Y68V

32.7±1.9

ND a

G109A

88.1±3.0

11.2±2.1

G109L

34.5±2.3

6.5±0.62

G109I

43.2±3.4

ND a

G109P

72.8±5.3

16.3±0.92

G109F

ND

5.4±0.23

G109V

24.7±2.6

ND

31



Page 32 of 40

Table 2. The t1/2 and Tm values of the wild type and variant enzymes with the absence or presence of 1 mM Co2+. Enzyme Wild-type Y68I Y68F G109A G109P Y68I/G109P

t1/2 (min) 43.3 38.5 21.6 31.9 92.4 78.3

Tm (°C) 54.6 53.5 51.1 51.7 59.3 55.4

Enzyme (Co2+) Wild-type Y68I Y68F G109A G109P Y68I/G109P

32


t1/2 (min) 156.0 122.8 73.2 112.1 327.4 264.0

Tm (°C) 61.1 60.3 57.5 59.4 66.7 63.5

Page 33 of 40


Table 3. Kinetic parameters of the bioconversion of D-fructose and D-tagatose by the wild-type and variant enzymes with substitutions at positions 68 and 109. Enzyme

kcat (min-1)

Km (mM)

Kcat/km (min-1 mM-1)

D-Fructose D-Tagatose D-Fructose D-Tagatose D-Fructose

D-Tagatose

Wild-type

59.8±8.2

339±6

3543±43

284±12

59.2±2.9

0.84±0.6

Y68I

46.05±7.5

1223±26

3764±54

123±5.6

81.7±3.2

0.10±0.012

Y68F

72.3±4.2

1645±14

2935±61

93±8

40.6±4.6

0.056±0.009

G109A

68.2±8.2

845±19

2543±49

115±13

37.3±2.9

0.13±0.015

G109P

86.1±1.2

674±12

2656±87

165±12

30.9±1.2

0.24±0.023

Y68I/G109P

49.1±1.2

942±10

3620±74

176±9

73.7±6.2

0.19±0.013

33



Fig. 1 Multiple sequence alignment of DPEases from various microorganisms. The origins of DPEase enzymes with the GenBank accession numbers as follows: Clce-DPEase (C. cellulolyticum DPEase, ACL75304), ClboDPEase (C. bolteae DPEase, EDP19602), Agtu-DPEase (A. tumefaciens DPEase, AAK88700.1), Clsc-DPEase (C. scindens DPEase, EDS06411.1), Clsp-DPEase (Clostridium sp. DPEase, YP_005149214.1), Desp-DPEase (Desmospora sp. DPEase, WP_009711885.1), and Rusp-DPEase (Ruminococcus sp. DPEase, ZP04858451). 201x140mm (150 x 150 DPI)


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Fig. 2 Effects of different pH values on the activities and stabilities of the wild-type C. bolteae DPEaseand its variants. (A) Optimum pH of the wild-type enzyme and the variant enzymes. The enzymatic activities were determined at 55 °C for 5 min in buffers with different pH (5.0 to 9.0) values, in the presence of 0.4 mM Co2+. (B) The pH stability of the enzymes was tested by pre-incubating the purified enzymes in solutions with various pH values (pH 5.0 to 9.0) at 4 °C for 2 hours. All of the assays were performed in triplicate. 373x130mm (150 x 150 DPI)



Fig. 3 Effects of different temperatures on the activity and thermostability of the wild-type C. bolteae DPEase and Y68I/G109P variants. (A) Optimum temperature of the wild-type and variant enzymes. The enzyme activities were determined in 50 mM sodium phosphate buffer (pH 7.0) at between 40 and 80 °C for 5 min, in the presence of 0.4 mM Co2+. All of the assays were performed in triplicate. (B) The DSC datas of wild-type and Y68I/G109P variant enzymes with 1mM Co2+. 587x203mm (96 x 96 DPI)


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Fig. 4 D-Psicose production from D-fructose by wild-type and Y68I/G109P enzymes. All the reactions were performed at pH 7.0 and 55 °C, containing 0.5 µM enzyme,1 mM Co2+ and 500 g/L of D-fructose. Results are the mean values of three experiments. 215x150mm (150 x 150 DPI)



Fig. 5 Three-dimensional model of predicted by SWISS-MODEL. (A) A model structure of C. bolteae DPEase using the crystal structure of C. cellulolyticum DPEase as template. (B) Superimposition of the monomer of C. bolteae DPEase (green) and C. cellulolyticum DPEase (pink). (C) The superposition of active site from C. bolteae DPEase (yellow) and C. cellulolyticum DPEase (cyan). 705x209mm (96 x 96 DPI)


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Fig. 6 Putative active site of the wild-type C. bolteae DPEase (A) and the double-site Y68I/G109P variant DPEase (B). The hydrogen bonds of amino residues at 68 position were shown using green dotted lines. 321x160mm (150 x 150 DPI)



TOC Graphic 405x312mm (96 x 96 DPI)


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Improving the Thermostability and Catalytic Efficiency of the d-Psicose

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