Probing the Role of Two Critical Residues in Inulin Fructotransferase

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Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-producing) Thermostability from Arthrobacter sp. 161MFSha2.1 Shuhuai Yu, Xiao Wang, Tao Zhang, Bo Jiang, and Wanmeng Mu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02291 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 25, 2016

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Journal of Agricultural and Food Chemistry

Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-producing) Thermostability from Arthrobacter sp. 161MFSha2.1 Shuhuai Yu†, Xiao Wang†, Tao Zhang†, 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 Universtiy,

Wuxi, Jiangsu, 214122, China

Corresponding authors: *

(W.

Mu)

Phone:

+86

510

85919161.

Fax:

E-mail:[email protected].

1

ACS Paragon Plus Environment

+86

510

85919161.


1

Abstract

2

Inulin fructotransferase (IFTase) is an important enzyme that produces

3

di-D-fructofuranose 1,2':2,3' dianhydride (DAF III) which is beneficial for human

4

health. Present investigations mainly focus on screening and characterizing IFTase,

5

including catalytic efficiency and thermostability which are two important factors for

6

enzymatic industrial applications. However, few reports aimed to improve these two

7

characteristics based on the structure of IFTase. In this work, a structural model of

8

IFTase (DFA III-producing) from Arthrobacter sp. 161MFSha2.1 was constructed

9

through homology modeling. By analyzing this model, two residues, Ser-309 and

10

Ser-333, may play key roles in the structural stability. Therefore, the functions of the

11

two residues were probed by site-directed mutagenesis combined with the Nano-DSC

12

method and assays for residual activity. In contrast to other mutations, single mutation

13

of serine 309 (or serine333) to threonine did not decrease the enzymatic stability,

14

whereas double mutation (serine 309 and serine333 to threonine) can enhance

15

thermostability (approximately 5 °C). The probable mechanisms for this enhancement

16

were investigated.

17 18

Key words: Di-D-Fructofuranose 1,2':2,3' dianhydride (DFA III); Inulin; Inulin

19

fructotransferase (IFTase); Homology modeling; Thermostability

2


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20

Introduction

21

Fructose is usually a critical carbohydrate for metabolism in organisms. However,

22

this monosaccharide is often stored in the form of fructans in 15% of flowering plants

23

as well as in a wide range of bacteria or fungi 1. In addition to serving as an energy

24

storage reservoir

25

environments 3. Inulin and levan are two primary fructans that consist of linear β-2,1-

26

and β-2,6-linked fructosyl residues, respectively. They display physiological functions

27

for human health to some extent. Specifically, inulin is considered as functional food

28

and feed because of its prebiotic properties 4. For example, it selectively enhances the

29

growth of bifidobacteria and lactobacilli 5. Moreover, calcium intake can be

30

significantly promoted by ingestion of inulin-type fructans

31

principally

32

sucrose:sucrose

33

1-fructosyl-tansferase (1-FFT). During the synthetic process, 1-SST catalyzes the

34

formation of 1-kestose, a trisaccharide, by transferring one sucrose fructosyl residue

35

onto other sucrose molecules. Subsequently, chain elongation is achieved by 1-FFT 8, 9.

36

In contrast, bacterial inulin is mainly biosynthesized by inulosucrase, which belongs

37

to glycosyl hydrolase (GH) family 68 based on the classification of the

38

‘carbohydrate-active enzymes (CAZy)’ database (http://www.cazy.org/)

39

degradation of fructans to small molecular carbohydrates facilitates energy acquisition.

40

This

41

β-fructofuranosidase (3.2.1.26) 14 and exo-inulinase (E.C. 3.2.1.7) 15, which hydrolyze

1, 2

, fructans also function as protectants against various

biosynthesized

process

by

two

distinct

1-fructosyl-transferase

usually

needs

various

enzymes

(1-SST)

enzymes.

6, 7

. In nature, inulin is

in

plants,

and

For

3


inulin,

including

fructan:fructan

it

10-13

. The

involves


16

42

inulin into fructose; endo-inulinase (EC. 3.2.1.7)

43

fructo-oligosaccharides; and inulin fructotransferases (IFTases), which decompose

44

inulin into di-D-fructofuranose 1,2':2,1' dianhydride (difructose anhydride I, DAF I)

45

(EC 4.2.2.17 for DFA I-forming IFTase)

46

dianhydride (difructose anhydride III, DAF III) (EC 4.2.2.18 for DFA III-forming

47

IFTase) 18, 19.

17

, which converts inulin into

or di-D-fructofuranose 1,2':2,3'

48

Among these inulin products, DFA III, which consists of two fructose molecules

49

with two reciprocal glycosidic linkages 20, has attracted increasing attention from the

50

scientific and commercial community because of its promising physiological

51

functions. For example, due to its low calories (52% sweetness but only 1/15 energy

52

of sucrose)

53

effect of accelerating calcium or iron absorption in the intestines 22, functional foods

54

to treat osteoporosis and iron-deficiency anemia can be made by supplementation

55

with DFA III. Considering these benefits for human health, DFA III has been

56

industrially manufactured using commercially available Arthrobacter sp. H65-7

57

IFTase (DFA III-forming) by the Shimizu Factory (Tokachi District, Hokkaido, Japan)

58

of the Nippon Beet Sugar Mfg. Co., Ltd. since 2004

59

III-containing commodities have been on the market since 2004 and in drugstores and

60

convenience stores since 201124.

21

, it can be used as an ideal sucrose substitute. Additionally, given the

23

. Moreover, in Japan, DFA

61

Aside from the aforementioned functional research and commercial application of

62

DFA III, the synthesis of DFA III has been extensively investigated. Presently, the

63

major manufacturing approach is through biosynthesis with microorganisms or IFTase. 4


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64

Wang et al. summarized the microorganisms and IFTases that produce DFA III up to

65

2015 25. Furthermore, two novel IFTases (DAF III-producing) were elaborated on by

66

Haraguchi et al.

67

microorganisms or enzymes. In contrast, few reports have examined improving

68

enzymatic activity or thermostability, which are two important factors of industrial

69

enzymes. Although it was reported that catalytic behavior and thermal stability of

70

IFTase can be enhanced by applying high hydrostatic pressure 28, 29, few investigations

71

have examined increases in activity or thermostability based on protein structure. In

72

2007, the three-dimensional crystallographic structure of Bacillus sp. snu-7 IFTase

73

(BsIFTase) was first resolved by Jung et al., which revealed that the enzyme is a

74

homotrimer

75

characteristic enhancement on the basis of the BsIFTase structure. In this work, we

76

constructed a model of IFTase from Arthrobacter sp. 161MFSha2.1 (AsIFTase) based

77

on the BsIFTase structure (AsIFTase, which is a homotrimer as BsIFTase, has been

78

fully characterized in our previous investigation 27). In this model, two serine residues,

79

Ser-309 and Ser-333, showed potential critical roles in stabilizing the structure.

80

Therefore, a rational design was implemented to probe the functions of these two

81

residues and to enhance the thermostability of AsIFTase, which may make this IFTase

82

to be an ideal industrial enzyme.

26

and Wang et al.

27

in 2015. These studies focused on screening

30

. However, there has been no further exploration on enzymatic

83 84

Materials and methods

85

Chemicals, reagents, and strains. The gene cloning agents were obtained from 5



86

Sangon Biotech Co., Ltd. (Shanghai, China). Electrophoretic reagents were obtained

87

from Bio-Rad (Hercules, CA, USA). The Ni2+-chelating affinity chromatography resin

88

was purchased from GE (Uppsala, Sweden). Isopropyl-β-D-1-thiogalactopyranoside

89

(IPTG) for induction was from Sigma (St. Louis, Mo, USA). Inulin (Orafti HP,

90

molecular weight is approximately 5,000 Da) was purchased from BENEO-Orafti NV

91

(Tienen, Belgium). Other chemicals were at least of analytical grade and were

92

obtained from Sigma (St. Louis, MO, USA) or Sinopharm Chemical Reagent

93

(Shanghai, China).

94 95

Escherichia coli (E. coli) BL21 (DE3) and DH5α cells were obtained from Sangon Biological Engineering Technology and Services (Shanghai, China).

96 97

Molecular modeling. The homology construction of the structural models was

98

performed with the SWISS-MODEL server (http://www.expasy.ch/swissmod/

99

SWISS-MODEL.html) 30-33 using the BsIFTase crystal structure (PDB ID: 2INV) 30 as

100

a template. The structure energy minimization was implemented by Discovery Studio

101

software (Accelrys, CA, USA). Subsequently, the quality of the AsIFTase and mutant

102

models was checked by the VERIFY_3D Server 34-36. The stereochemical quality was

103

validated by Coot with its Ramachandran plot module

104

rendered and presented with the Pymol Molecular Graphics System.

37

. The structures were

105 106

Mutagenesis, heterologous expression, and purification. Based on analysis of the

107

AsIFTase model, residues Ser-309 and Ser-333 may be important for enzyme stability. 6


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108

Hence, a rational design of the two critical residues was implemented, and

109

site-directed mutagenesis was performed by one-step PCR methods using a TaKaRa

110

MutantBEST Kit (TaKaRa, Dalian, China). In total, eight single-point mutants

111

(S309A, S309C, S309F, S309T, S333A, S333C, S333F, and S333T) and four double

112

mutations (S309A/S333A, S309C/S333C, S309F/S333F, and S309T/S333T) were

113

designed (Table 1). The pET-22b(+) plasmid constructed in a previous work

114

used as a template with the primers shown in Table 1. According to the TaKaRa

115

MutantBEST Kit protocol, the 50 µL PCR reaction system contained 0.01 – 1.0 ng of

116

plasmid template, 10× pyrobest Buffer II, 0.25 µL of pyrobest DNA polymerase

117

(TaKaRa, Dalian, China) (5 U µL-1), 1 µL of each primer (20 µM), 4 µL of dNTP

118

mixture (2.5 mM for each). Finally, ddH2O was used to bring the reaction to the final

119

volume. The PCR reaction was performed with following program: predenaturation at

120

94 °C for 1 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30

121

s, and elongation at 72 °C for 5 min; and a final extension step at 72 °C for 10 min.

122

Subsequently, a reaction containing 50 µM DNA fragment, 2 µL of 10× blunting

123

buffer, 1 µL of Blunting Kination enzyme mix, and a suitable amount of ddH2O,

124

which was used to bring the final volume to 20 µL, was used to phosphorylated the

125

5′-ends of the amplified fragments. Finally, 5 µL of DNA ligation solution I was

126

added to the above reaction system for self-ligation at 16 °C for 1 h.

127 128

27

was

Induction, expression, and purification of the AsIFTase mutants were carried out similarly to that of AsFTase in our previous work 27.

129 7



130

The effect of temperature on enzyme activity and thermostability. The assay

131

methods and unit definition of enzymatic activity were reported in our previous

132

work27. Temperatures were from 30 to 80 °C.

133

The thermostability of the enzyme was investigated as follows. First, because

134

Differential Scanning Calorimeters (Nano DSC, TA Instruments, New Castle, DE) can

135

sensitively capture the change in energy in protein folding and unfolding processes,

136

the melting temperature (Tm) was used to reflect the thermostability of the enzyme. At

137

3 atmospheric pressures (approximately 304 kPa), the sample (0.1 mg mL-1) was

138

detected at a heating rate of 1 °C min-1 from 30 to 100 °C. Second, referring to our

139

previous work 27, the thermostability was inspected by detecting the residual activity

140

after incubating the enzymes at 55, 60, 70, and 80 °C for 240 min. Thirdly, to

141

determine the enzymatic thermostability, which was indicated by the half-life time of

142

heat denaturation (t1/2), the enzymes were incubated at 55 °C for different times and

143

the residual activities were measured at 55 °C and pH 5.5.

144 145

Results

146

Prediction of the AsIFTase structure

147

Because BsIFTase shared high sequence identity (83%) with AsIFTase, the 30

148

crystallographic structure of BsIFTase (PDB ID: 2inv)

was selected as a template

149

for homology modeling. After structure energy minimization, the VERIFY-3D module

150

demonstrated that 97% of the residues had an average 3D-1D score >= 0.2, which was

151

higher than the minimal requirement (80%). Subsequently, the Ramachandran plot 8


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152

(Fig. S1) showed that the total percentage, including preferred regions (93.7%) and

153

allowed regions (3.86%), was rather high (more than 95%). These results indicated

154

that the overall quality of the model was acceptable.

155

As shown in Fig. 1A, the monomeric model of AsIFTase has a right-handed parallel

156

β-helix, which is similar to BsIFTase. In addition, superimposition of the overall

157

structures showed that the two structures are similar (Fig. 1B). In both structures,

158

hydrogen bonds from residue Ser-309 protrude into the central axis forming a triangle.

159

Although many residues in one subunit formed hydrogen bonds with the second

160

subunit, the residue Ser-309 in one subunit can simultaneously form hydrogen bonds

161

with other two subunits, which displayed a unique intersubunit contact.

162 163

Gene cloning, heterologous expression, and purification of the mutants

164

Given the specificity of the triangular pattern (hydrogen bonds) between the

165

Ser-309 residues, which may play a critical role in the stability of the structure,

166

site-directed mutagenesis was implemented to investigate the functions of this site.

167

Additionally, Ser-333, which is above Ser-309 (Fig. 2A), had considerable probability

168

to form this triangular pattern through mutation, which would also contribute to the

169

stability of the three-dimensional structure. Hence, mutation of Ser-333 was also

170

performed. Eight single-point mutants (S309A, S309C, S309F, S309T; S333A, S333C,

171

S333F, and S333T) were designed for Ser-309 and Ser-333, respectively (Table 1).

172

Four double mutations, including S309A/S333A, S309C/S333C, S309F/S333F, and

173

S309T/S333T, were also made. After cloning the genes and heterologous expression,

174

the Ser-309 and Ser-333 mutants were purified as single strands from a gel with a

175

molecular mass of approximately 42 kDa (Fig. 3), which indicated that all the mutants

176

expressed correctly in the E. coli system. 9



177 178

The effect of mutation on the enzyme activity and thermostability

179

Mutation of Ser-309 and Ser-333 may change the reaction temperature and

180

stability of the three-dimensional structure. Hence, the dependence of enzyme activity

181

on temperature was determined to identify the optimum temperature and

182

thermostability. As shown in Table 2, compared with the optimum reaction

183

temperatures (Topt) for wild-type AsIFTase (55 °C), the Topt values for the

184

phenylalanine (Phe), cysteine (Cys), and alanine (Ala) mutants decreased by 5 °C,

185

while those for the threonine (Thr) mutants were invariable. Based on the mutant Topt

186

values, the relative enzyme activities were more than 90%, except for the two double

187

mutants (S309A/S333A and S309C/S333C) (Table 2).

188

For thermostability, in comparison with wild-type AsIFTase via the Nano-DSC

189

method (Fig. 4), the Tm value of the Phe, Ser, and Ala mutants successively decreased,

190

while the Tm values increased by approximately 2 and 5 °C for the single and double

191

Thr mutants, respectively (Table 2). Simultaneously, after incubation at 55, 60, 70,

192

and 80 °C for 240 min, the results of the residual activities can be summarized into

193

three facets. First, the thermostability of all the enzymes, which was indicated by

194

residual activities, gradually declined with the increase in temperature (Fig. 5).

195

Second, in comparison with wild-type AsIFTase, the thermostability of the Ala, Cys,

196

and Phe mutants decreased. This downward trend was more obvious with the increase

197

in temperature. Moreover, the falling velocity of the Phe mutants was slightly slower

198

than that of the Ala and Cys mutants. Third, in contrast to other mutants, there were 10


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199

very slight differences in the thermostability between the wild-type enzyme and the

200

single Thr mutants. In contrast, the stability of the double Thr mutant was obviously

201

improved, and this improvement was more remarkable with increases in temperature.

202

Lastly, in comparison with wild-type AsIFTase (shown in Table 2), the t1/2 at 55 °C

203

was enhanced by approximately 2 and 4 h for the single and double Thr mutants,

204

respectively, while it was reduced by at least 5 h for the other mutants.

205 206

Homology modeling of the mutants

207

The homology modeling process and quality check was similar to that of

208

wild-type AsIFTase. The final mutant models were acceptable and quite similar to that

209

of wild-type AsIFTase (data not shown). However, the Ser-309 and Ser-333 linkages

210

with nearby residues were varied (Fig. 6), which may contribute to the differences in

211

the aforementioned thermostability of the mutants (detailed changes are analyzed in

212

the Discussion section).

213 214

Discussion

215

In general, it is difficult to obtain three-dimensional coordinates for each enzyme.

216

However, because the tertiary structure is determined by the primary structure, it is

217

possible to obtain enzymatic structures through homology modeling. Usually, the

218

constructed models are reliable when enzymes share finely high sequence identity

219

with proteins having known structures. These models allow enzymes to be rationally

220

designed and modified.

221

In this work, the AsIFTase monomer displayed a typical right-handed parallel 11



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222

β-helix (Fig. 1A) and the root mean square deviation (RMSD) of the Cα atoms

223

between the two structures by superimposition (Fig. 1B) was 0.063 (1150 to 1150

224

atoms), which indicated that the two structures were quite similar. Moreover, it

225

revealed that residue Ser-309 forms a unique triangular hydrogen bond linkage, which

226

may play a critical role in stabilizing the structure. Likewise, BsIFTase also displayed

227

this pattern. However, the linkages between Ser-309 and nearby residues were

228

different. As seen in Fig. 2A, in addition to the triangular linkage, Ser-309 also

229

formed a hydrogen bond (3.2 Å) with Ser-308 from a neighboring subunit, which may

230

enhance the structural stability. Moreover, Ser-333, which also bonds to Ser-308 (2.6

231

Å) from the same subunit, formed a weak hydrogen bond with Ser-309 (3.4 Å).

232

Therefore, Ser-333 may act like a crab claw that indirectly stabilizes the overall

233

structure through clamping Ser-309 and Ser-308 and consolidating the linkage

234

between these two residues. This clamp role of Ser-333 is similar to Asp-233 in

235

BsIFTase for stability purposes, though Asp-233 stabilizes the substrate rather than the

236

three-dimensional structure

237

Ser-309, and Ala-333 (corresponding to Ser-308, Ser-309, and Ser-333 in AsIFTase,

238

respectively) were absent (Fig. 2B). Therefore, in comparison with AsIFTase, the

239

lower thermostability (103-min half-life at 60 °C for BsIFTase

240

half-life at 80 °C for AsIFTase 27) may be partly ascribed to this absence. As shown in

241

Fig. S2, all the IFTases (DFA III-forming) with GeneBank accession had 82%

242

sequence identity. This considerably high identity indicated that IFTases (DFA

243

III-forming) may have similar structures. Fig. S2 shows that residues corresponding to

30

. However, in BsIFTase, linkages between Thr-308,

12


38

and 240-min

Page 13 of 34


244

the BsIFTase active center and residues corresponding to Ser-308 and Ser-309 in

245

AsIFTase were highly conserved, while residues corresponding to Ser-333 in

246

AsIFTase were variable. Like Ala-333 in BsIFTase, this variation may lead to a

247

decrease in structural stability.

248

Due to the importance of Ser-309 and Ser-333 from a previous analysis, these sites

249

were mutated to four different amino acids. Mutation with a smaller Ala and a larger

250

Phe were designed to verify the importance of Ser-309 and Ser-333. The Cys mutant

251

was designed to improve the thermostability because disulfide bond which is firmer

252

than hydrogen bonds may be formed between cysteines. Thr, which is similar to Ser,

253

was introduced to form triangular hydrogen linkages and to reinforce the stability of

254

the structure. As seen in Fig. 3, the individual mutant protein bands on the SDS-PAGE

255

gel were approximately 42 kDa. This agreed well with the molecular mass of

256

wild-type AsIFTase in our previous work 27. Wild-type AsIFTase was also purified and

257

the SDS-PAGE result was similar to our previous work (data not shown).

258

After purification, the effect of temperature on enzyme activity was investigated.

259

The activity of all the enzymes was determined at their respective Topt values (50 or

260

55 °C). Table 2 showed that the relative activities of most of the mutants were more

261

than 90%, though the double Ala and Cys mutants displayed a slightly lower relative

262

activity (88 and 84%, respectively), which indicated that mutation of Ser-309 and

263

Ser-333 had little effect on activity. These data can be explained by the structure. Due

264

to the considerable sequence identity and structural similarity between AsIFTase and

265

BsIFTase, we can infer that the AsIFTase active center is located at a

266

monomer-monomer interface like BsIFTase, which means that Ser-309 and Ser-333

267

were far away from this putative active center. Therefore, Ser-309 and Ser-333 had no

268

direct role in catalyzing or binding substrate, which indicated that the effect of 13



269

mutation on activity was not obvious.

270

Thereafter, the thermostability of the mutants was investigated. First, the

271

thermostability was determined by Nano-DSC (Fig. 4). As shown in Table 2, in

272

comparison with the wild-type enzyme (87.04 °C), the Tm values for the Ala, Cys, Phe

273

mutants dropped by 13 to 25 °C. This result indicated that mutation of the Ser-309 or

274

-333 residues to Ala, Cys, and Phe decreased the enzymatic stability. Moreover, the

275

Tm of Phe, Cys, and Ala displayed a successive downtrend, which suggested that the

276

Phe mutant was slightly more stable than the Ala and Cys mutants. Second, the

277

residual activities were assayed to determine the thermostability. Fig. 5 showed that

278

the residual activities of all the enzymes decreased with an increase in temperature.

279

This phenomenon conformed to common regularity due to the unfolding of protein at

280

higher temperatures 39. Moreover, at low temperatures (55 °C), enzymes including the

281

Ala, Cys, and Phe mutants maintained more than 74% of their residual activity (Fig.

282

5). However, at higher temperatures (60 to 80 °C), the residual activities of the Ala,

283

Cys, and Phe mutants substantially dropped. Interestingly, the residual activities of the

284

Phe mutants were always higher than those of the Ala and Cys mutants at the same

285

temperature. This indicated that the Phe mutants were more stable than Ala and Cys

286

mutants, which agreed well with the Nano-DSC results. In contrast to the other

287

mutants, the residual activities of the single Thr mutants slightly changed compared

288

with the wild-type enzyme at the same temperature, while the double Thr mutation

289

enhanced the residual activities. This indicated that the mutation of Ser to Thr at

290

amino acids 309 and 333, respectively, maintained or enhanced the structural stability.

291

This enhancement was also in accordance with the Nano-DSC results. The enhanced

292

effect of the double Thr mutant was more remarkable, especially at higher

293

temperatures. For example, at 55 °C, the wild-type enzyme, S309T, S333T, and 14


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294

double mutants maintained approximate residual activities (90, 90, 88, and 91%

295

respectively). However, at 80 °C, their residual activities were 52, 53, 56, and 62% of

296

residual activities, respectively. These changes in residual activity suggest that

297

wild-type AsIFTase and the Thr mutants maintained their stability at low temperatures,

298

and the double Thr mutant was more stable at higher temperatures. Lastly, the assay

299

for t1/2 at 55 °C was implemented (Table 2). In comparison with wild-type AsIFTase,

300

the t1/2 values of the Thr mutants increased by 2 to 4 h, while those of the other

301

mutants decreased by at least 5 h. This also validated that the thermostability of the

302

double Thr mutants was more stable than those of the wild-type enzyme or other

303

mutants.

304

Herein, the increase or decrease in thermostability due to the mutations can be

305

explained by changes in the linkages between the residues. As mentioned above, the

306

triangular linkages formed by Ser-309 residues and linkages between Ser-309 and

307

S308 from neighboring subunits may play a critical role in stabilizing the structure

308

(Fig. 2A). However, these linkages were broken by mutating Ser to Ala, Cys, and Phe

309

(Fig. 2A–2C), which led to the decrease in thermostability. In the Phe mutant,

310

although these linkages were absent, an edge-to-face (3.6 Å) and a quite weak offset

311

face-to-face (4.1 and 4.2 Å) π-π stacking interaction probably formed between the Phe

312

residues (Fig. 6C). That is why the thermostabilities of the Phe mutants decreased

313

slower than those of the other mutants. Furthermore, the mutation of Ser to Cys was

314

originally designed to introduce disulfide bonds between the 309 residues; however,

315

no bond (even hydrogen bond) was formed (Fig. 6B). In contrast to the Ala, Cys, and

316

Phe mutants, the mutation of Ser-309 to Thr-309 did not change the interaction ways

317

of these residues (Fig. 6D). Simultaneously, the linkages between Thr-309 and

318

Ser-308 (or Thr-309) were slightly strengthened (3.2 to 3.1 Å between Thr-309 and 15



Page 16 of 34

319

Ser-308 and 2.9 to 2.8 Å between Thr-309 and Thr-309). This is why the

320

thermostability of the Thr-309 mutant did not decrease as that of Ala, Cys, and Phe

321

mutants. For the Thr-333 mutant, a new triangular hydrogen bond linkage was formed

322

between the Thr-333 (Fig. 6E), which may contribute to the maintenance of

323

thermostability. As mentioned above, the clamp-like hydrogen bonds made by residue

324

333 with S308 (or 309) may indirectly stabilize the structure. However, this clamp

325

was lost in the Thr-333 mutant. The slight rather than substantial enhancement at

326

higher temperature in the thermostability of the Thr-333 mutant can be ascribed to this

327

loss. Lastly, the improvement in stability for the double Thr mutant may be the

328

comprehensive result of Thr-309 and Thr-333 (Fig. 6F).

329

In summary, based on homology modeling and site-directed mutagenesis, two

330

residues, Ser-309 and Ser-333, were proven to be important for AsIFTase

331

thermostability. Thermostability was improved by approximately 5 °C through double

332

mutating Ser to Thr, which can be ascribed to the changes in the linkages between

333

Ser-308, Ser-309, and Ser-333. Given the perfect characterization of AsIFTase

334

reported in our previous work

335

may make this enzyme ideal for developing DFA III products.

27

, the improvement in thermal stability in this work

336 337

Author information

338

Corresponding author

339

*

340

E-mail:[email protected].

(W.

Mu)

Phone:

+86

510

85919161.

Fax:

341

16


+86

510

85919161.

Page 17 of 34


342

Funding Information

343

This work was supported by the NSFC Project (Nos. 31371788 and 21276001),

344

the 863 Project (No. 2013AA102102), the Support Project of Jiangsu Province (No.

345

BK20130001 and 2015-SWYY-009), and the project of outstanding scientific and

346

technological innovation group of Jiangsu Province (Jing Wu).

347 348 349

Abbreviations used IFTase,

inulin

fructotransferase;

DFA III,

di-D-fructofuranose

1,2':2,3'

350

dianhydride; AsIFTase, Arthrobacter sp. 161MFSha2.1 IFTase; BsIFTase, Bacillus sp.

351

snu-7

352

isopropyl-β-D-1-thiogalactopyranoside;

353

polyacrylamide gel electrophoresis; LB medium, Luria-Bertani medium; DSC,

354

differential scanning calorimetry

IFTase;

HPLC,

high-performance

liquid

SDS-PAGE,

chromatography; sodium

dodecyl

IPTG, sulfate;

355 356 357

Competing interests The authors declare that no competing interests exist.

358 359 360 361

Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors.

362 363

Author Contributions 17



364

SY, XW, TZ, BJ, and WM conceived and designed the experiments. SY and XW

365

performed the experiments. SY, XW and WM analyzed the data. SY, XW, and WM

366

contributed reagents/materials/analysis tools. SY and WM wrote the paper.

367 368

Supporting information

369

Fig. S1. Ramanchandran plot of AsIFTase model.

370 371

Fig. S2. Multiple sequence alignments of IFTases (DFA III-forming).

372 373

References

374

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18. Tanaka, K.; Uchiyama, T.; Ito, A., Formation of di-d-fructofuranose 1,2′:2,3′

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（difructofuranose 1,2',2,3'-anhydride）. J. Res. Natl. Bur. Stand. 1940, 24, 181-204.

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26. Haraguchi, K., Difructose Dianhydride III Producing inulin fructotransferase

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33. Guex, N.; Peitsch, M. C.; Schwede, T., Automated comparative protein structure

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34. Bowie, J. U.; Luthy, R.; Eisenberg, D., A method to identify protein sequences

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37. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K., Features and development of

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ssion, and characterization of Bacillus sp. snu-7 inulin fructotransferase. J. Micr

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39. Bisswanger, H., Enzyme assays. Perspectives in Sci. 2014, 1, 41-55.

23



477

Figure legends

478

Fig. 1. AsIFTase models and structural alignment of AsIFTase and BsIFTase. (A)

479

Monomeric model of AsIFTase. β-sheets are delineated with cyan, yellow, and red

480

colors. The probable 310-helices and α-helix are colored in green. (B) Superimposition

481

of the AsIFTase model (green) onto the crystallographic structure for BsIFTase (PDB

482

ID: 2inv) (cyan). The Ser-309 residues from AsIFTase and BsIFTase protrude from the

483

core β-helices, forming three regular protrusions in the intersubunit channel along the

484

central axis. Hydrogen bonds between the three AsIFTase or BsIFTase Ser-309

485

residues form a triangle with 2.9 Å distances, which stabilize the overall structure.

486 487

Fig. 2. Hydrogen bonds formed between Ser-309, Ser-308, and Ser-333 in (A)

488

AsIFTase and (B) BsIFTase. The different colored dash lines represent hydrogen

489

bonds. The hydrogen bonds distances (angstrom) are labeled with black Arabic

490

numerals.

491 492

Fig. 3. SDS-PAGE analysis of the mutants. (A) Lane M: standard protein marker; lane

493

1 to 6: S309A, S309C, S309F, S309T, S309A/S333A, and S309C/S333C. (B) Lane M':

494

standard protein marker (the same to that of Lane M); Lane 1' to 6': S333A, S333C,

495

S333F, S333T, S309F/S333F, and S309T/S333T.

496 497

Fig. 4. Wild-type AsIFTase Nano-DSC results. The Tm corresponds to the peak

498

temperature. The mutant Nano-DSC profiles were similar to the wild-type AsIFTase 24


Page 24 of 34

Page 25 of 34


499

profile (data not shown).

500 501

Fig. 5. Thermostability of wild-type AsIFTase and the mutants. The enzymes were

502

incubated at 55, 60, 70, and 80 °C for 240 min. The residual activity was defined as

503

the percentage of initial enzymatic activity. Each experiment was carried out in three

504

replications ± standard deviation.

505 506

Fig. 6. The linkages between the Ser-308, Ser-309, and Ser-333 residues. Ser-308,

507

Ser-309, and Ser-333 are labeled pink, green, and orange, respectively. The dash

508

represents hydrogen bonds (cut-off was 3.3 Å) or distance between the residues. The

509

length of the dashed lines is labeled with black Arabic numerals. (A)-(F) represent

510

linkages of S309A, S309C, S309F, S309T, S333T, S309T/S333T, respectively.

25



Table 1. Primers for site-directed mutagenesis. The underlined sequences represent mutated codons. Double mutation was implemented by mutating Ser-309 (Ser-333) after the successful single mutation of Ser-333 (Ser-309). Mutant sites S309A-Forward S309C-Forward S309F-Forward S309T-Forward Ser-309-Reverse S333A-Forward S333C-Forward S333F-Forward S333T-Forward Ser-333-Reverse

Oligonucleotides（（5′-3′）） CGTATCGGCGAACCGGTTTCAGGGCTT CGTATCGTGCAACCGGTTTCAGGGCTTC CGTATCGTTCAACCGGTTTCAGGGCTTC CGTATCGACTAACCGGTTTCAGGGCTTC GAGCAGCGGTTACAGCCGTTGAACT ATCACGGCGAATCACTTCCGCAGGG ATCACGTGCAATCACTTCCGCAGGG ATCACGTTCAATCACTTCCGCAGGG ATCACGACTAATCACTTCCGCAGGG GAGGTTTTCCTTGCATCCATTCAG

26


Page 26 of 34

Page 27 of 34


Table 2. The results of effect of temperature on enzyme activity. Topt represents optimum temperature. The relative activity of the mutants was defined as a percentage of the wild-type enzyme activity. The melting temperatures (Tm) of the mutants using the Nano-DSC method were compared with the wild-type enzyme (87.04 °C was defined as 0, and the other Tm values were calculated by subtracting 87.04 °C). The t1/2 represents the half-life time of enzymatic heat denaturation. The data is the mean of three replications ± standard deviation.

wild S309A S309C S309F S309T S333A S333C S333F S333T S309A/333A S309C/333C S309F/333F S309T/333T

Topt (°C) 55 50 50 50 55 50 50 50 55 50 50 50 55

Activity (%) 100 ± 2.3 95.80 ± 3.2 94.90 ± 2.1 102.60 ± 4.3 103.50 ± 3.9 98.60 ± 2.3 95.70 ± 4.1 97.40 ± 3.7 92.40 ± 2.5 88.20 ± 3.9 84.50 ± 4.4 96.40 ± 2.9 91.80 ± 3.1

Tm (°C) 0 (87.04) ± 0.4 - 17.88 ± 1.3 - 16.8 ± 0.9 - 15.83 ± 1.2 + 2.89 ± 1.5 - 18.83 ± 2.0 - 15.71 ± 1.1 - 13.29 ± 2.3 + 2.16 ± 3.0 - 25.88 ± 2.8 - 23.7 ± 1.4 - 19.52 ± 2.8 + 5.05 ± 1.0

27


t1/2 (h) 20.7 ± 2.0 11.5 ± 1.1 12.8 ± 0.9 11.0 ± 1.3 22.5 ± 1.8 10.5 ± 0.9 12.6 ± 1.3 15.0 ± 1.1 22.0 ± 0.8 8.3 ± 0.9 6.8 ± 0.7 10.0 ± 0.9 24.0 ± 1.3


Fig. 1 87x50mm (300 x 300 DPI)


Page 28 of 34

Page 29 of 34


Fig. 2 138x70mm (300 x 300 DPI)



Fig. 3 111x46mm (300 x 300 DPI)


Page 30 of 34

Page 31 of 34


Fig .4 286x199mm (300 x 300 DPI)



Fig. 5 287x201mm (300 x 300 DPI)


Page 32 of 34

Page 33 of 34


Fig. 6 549x348mm (300 x 300 DPI)



For TOC only 241x178mm (150 x 150 DPI)


Page 34 of 34

Probing the Role of Two Critical Residues in Inulin Fructotransferase

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