Structural and Functional Basis of Difructose Anhydride III Hydrolase

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Structural and functional basis of difructose anhydride III hydrolase, which sequentially converts inulin using the same catalytic residue Shuhuai Yu, Hui Shen, Yuanyuan Cheng, Yingying Zhu, Xu Li, and Wanmeng Mu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b02424 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 12, 2018

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ACS Catalysis

Structural and Functional Basis of Difructose Anhydride III Hydrolase, Which Sequentially Converts Inulin Using the Same Catalytic Residue

Shuhuai Yu 1 , Hui Shen 2 , Yuanyuan Cheng 1 , Yingying Zhu 1 , Xu Li 2,* , Wanmeng Mu 1,3,*

1

State Key Laboratory of Food Science and Technology, Jiangnan University,

1800 Lihu Avenue, Wuxi, Jiangsu 214122, China 2

Hefei National Laboratory for Physical Sciences at Microscale and School of

Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China 3

International Joint Laboratory on Food Safety, Jiangnan University, 1800 Lihu

Avenue, Wuxi, Jiangsu 214122, China

Shuhuai Yu and Hui Shen contributed equally to this work. *Correspondence and requests for materials should be addressed to X.L. ([email protected]) or to W.M. ([email protected]).

1 ACS Paragon Plus Environment

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1

Abstract

2

Fructan inulin is composed of polymeric fructoses linked by O-glycosidic bonds,

3

and a variety of enzymes are involved in its decomposition to provide energy

4

for organisms. Specifically, inulin fructotransferase (IFTase) depolymerizes

5

inulin to difructose anhydride III (DFA-III). DFA-III was reported to be further

6

degraded by DFA-III hydrolyase (DFA-IIIase). This work reveals that the

7

structure of DFA-IIIase is a trimer, with each monomer displaying a right-

8

handed β-helix fold, which resembles IFTase except an extra lid covering the

9

active center. With this lid, DFA-IIIase is capable of converting inulin to DFA-

10

III (IFTase activity), in addition to hydrolyzing DFA-III using the same site

11

and reaction conditions. This unusual and unexpected sequential catalysis is

12

ascribed to the extremely conserved residues in the active center of IFTase and

13

DAF-IIIase and the protonated states of the catalytic residue that are regulated

14

by the opening and closure of the lid. This work paves the way for further

15

investigation of the metabolism of inulin in nature and provides a example of

16

sequential enzymatic catalysis.

17

Keywords: difructose anhydride (DFA); difructose anhydride III (DFA-III);

18

difructose anhydride hydrolase (DFAase); difructose anhydride III hydrolase

19

(DFA-IIIase); inulin; inulin fructotransferase (IFTase); sequential conversion


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ACS Catalysis

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Introduction

21

Fructan, storing energy for plants 1 or microorganisms 2 like starch, is composed

22

of polymeric fructoses linked by O-glycosidic bonds. Some natural occurring

23

fructan is terminated with a glucose, such as two important fructans inulin and

24

levan 3 . To provide energy for organisms, the large molecular fructans need to

25

be decomposed to the small-molecule sugars, a process that involves some

26

degrading enzymes. Fructan-degrading enzymes can be divided into three types

27

based on the catalytic reaction 4 . One type includes IFTase 5 and levan

28

fructotransferase 6-7 , which specifically liberates the difructose units from one

29

end of the molecular chain. These difructose units additionally form a new

30

anhydride linkage. Thus, the final product is a kind of difructose anhydride

31

(DFA). There are different types of DFA produced with fructans by specific

32

enzymes 8 . For example, levan can be degraded to DFA-IV (β- D -fructofuranose-

33

β- D -fructofuranose 2’,6:2,6’-dianhydride) by levan fructotransferase, while

34

inulin can be degraded to DFA-I (α- D -fructofuranose-β- D -fructofuranose

35

2’,1:2,1’-dianhydride)

36

2’,1:2,3’-dianhydride) by the corresponding IFTase 9 . To gain energy from

37

fructan for cell growth, DFAs are assimilated into cell and hydrolyzed by

38

difructose anhydride hydrolase (DFAase) to inulobiose. To date, only four

or

DFA-III

(α- D -fructofuranose-β- D -fructofuranose


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DFA-III hydrolases (DFA-IIIases) have been reported 10-13 . Although the

40

enzymatic mechanisms of degradation of the fructans inulin and levan into

41

DFAs has been well investigated 5,

42

inulobiose by DFAase is elusive. DFA-III has so many physiological functions

43

that its products have been on the market since 2014. Inulin to DFA-III, DFA-

44

III to inulobiose (Figure S1a), and inulobiose to fructose (by fructofuranosidase)

45

compose just one of inulin’s metabolic pathways. However, there are no reports

46

on physiological functions or even characteristics of inulobiose. We think that

47

the limited production of inulobiose limits its exploitation because there are

48

few reports on its synthesis.

14 ,

how DFAs are further degraded into

49

In this work, we identified a DFA-IIIase from Arthrobacter chlorophenolicus

50

A6 (termed as AcDFA-IIIase) and explored its catalytic mechanism by

51

resolving its structure. To the best of our knowledge, this is the first atomic

52

resolution structure of DFAase. Combination of the structural and functional

53

analysis provides a mechanism that clarifies one metabolic pathway of fructan

54

inulin in nature. In addition to the ability to hydrolyze DFA-III, AcDFA-IIIase

55

demonstrates an unexpected IFTase activity with the same reaction condition.

56

The mechanism of this unusual and interesting sequential catalysis was

57

investigated, and this reaction mode provides a distinctive example of


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ACS Catalysis

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sequential catalysis in enzymology. Moreover, the work also paves the way for

59

the investigation of the production, function, and application of inulobiose.

60 61

Results

62

Identification and characterization of AcDFA-IIIase. AcDFA-IIIase with

63

445 residues from Arthrobacter chlorophenolicus A6 shows 80% sequence

64

identity with reported DFA-IIIases and 35% - 50% identity with DFA III-

65

forming IFTases (Figure S1b). The enzyme was well expressed and purified

66

(Figure 1a). The molecular weight assayed by SDS-PAGE is approximately 47

67

kD a, which is consistent with the LC-MASS experiment result (Figure S2).

68

However, the native molecular mass of the enzyme assayed by gel filtration

69

experiment is approximately 139 kD a (Figure 1f), which indicates that the

70

enzyme is a kind of homotrimer in solution. The enzyme converts DFA-III to a

71

product corresponding to inulobiose (Figure 1b). Subsequently,

72

used to verify this as inulobiose. The NMR spectrum (Figure 1c) shows two

73

peaks at the chemical shift of 98.12 and 101.58 ppm, which specifically

74

correspond to those of anomeric carbon 2 (C-2) at the reducing end of 1-O-β-

75

D -fructofuranosyl- D -fructopyranose

76

fructofuranose, respectively. The chemical shift data are summarized in Table

and

13 C-NMR

was

1-O-β- D -fructofuranosyl- D -


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S1, and they agree well with those of reported inulobiose. Therefore, the

78

purified AcDFA-IIIase is a type of DFA-IIIase and two conformations

79

(pyranose and furanose) of inulobiose are produced.

80

AcDFA-IIIase was further characterized. Figure 1d and 1e show that the

81

enzyme has high activity around the pH of 6.5 and temperature of 55 °C.

82

Meanwhile, its activity rapidly decreases when the pH is more than 7.0 or when

83

the temperature is higher than 60 °C. The kinetic parameters were measured at

84

pH 6.5 and 55 °C, with its K m of 121.30 mM and k cat /K m of 4.61 s -1 mM -1 (Table

85

1).

86 87

Overall structure of AcDFA-IIIase. To explore the catalytic mechanism of AcDFA-

88

IIIase, we solved its structure. The details of the data collection and refinement statistics

89

are summarized in Table S2. There is only one molecule in one asymmetric unit in the

90

structure, differing with the homotrimer in solution (Figure 1f). The AcDFA-IIIase

91

structure, similar to the DFA-III-forming IFTase from Bacillus sp. snu-7 (BsIFTase)5,

92

forms a right-handed parallel β-helix fold (Figure 2a), which is frequently found in

93

enzymes associated with polysaccharides15-16. The Dali server was used to search for the

94

structures that are similar to the AcDFA-IIIase. The result shows that the AcDFA-IIIase is

95

homologous to the inulin fructotransferase from Bacillus sp. Snu-7 (PDB code 2INU) with

96

a Dali Z-score of 58.2 and an root mean square deviation (RMSD) of 0.9 Å for 394 Cα

97

atoms, the mannuronan C-5 epimerase from Azotobacter vinelandii (PDB code 2PYG)

98

with a Dali Z-score of 33.8 and an RMSD of 2.1 Å for 318 Cα atoms, and the tailspike 6 ACS Paragon Plus Environment

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ACS Catalysis

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endorhamnosidase from Shigella phage Sf6 (PDB code 2VBK) with a Dali Z-score of 30.5

100

and an RMSD of 2.5 Å for 328 Cα atoms. Specifically, there are 13 helical turns of a right-

101

handed coil, and the structure has an overall trihedral shape with mainly β-strands forming

102

the surface. AcDFA-IIIase shows homologous to pectate lyase (Pelc) with a Dali Z-score

103

of 17.7 and an RMSD of 2.6 Å for 222 Cα atoms. Similar to the turns of PelC, each turn of

104

AcDFA-IIIase consists of three β-strands joined by intervening loops apart from the N- and

105

C-terminal turns17-18. The first, second and third β-strands of each turn are designated PB1,

106

PB2 and PB3, respectively, with the following loops denominated T1, T2 and T3, just as

107

the designation in PelC (Figure 2b). All of the β-strands of PB1 form a long parallel β-

108

sheet, and the same structure is formed in PB2 and PB3. The β-strands of PB1 are almost

109

parallel to the β-strands of PB2, and the β-strands of both PB1 and PB2 are approximately

110

perpendicular to the β-strands of PB3. In the N-terminal turn, the PB1 β-strand of turn 1 is

111

substituted by a long α-helix followed by a long loop, T1. In the two C-terminal turns, turn

112

12 and 13, there are only two and one β-strands, respectively, which form two smaller turns

113

(Figure 2a). The T2 loop of turn 2 and the T3 loops of turns 2, 3, 9, 10 and 11 protrude

114

from the core parallel β-helix, forming a relatively irregular protrusion on one side of

115

AcDFA-IIIase. The T3 loop of turn 11, which has an α-helix, is particularly long, extending

116

almost half of the length of the structure. Similar to other β-helix proteins, the interior of

117

AcDFA-IIIase is closely packed with hydrophobic side chains along the sheet19.

118

Specifically, mainly Val, Leu, and Ile, located at the equivalent positions of each turn, are

119

stacked between neighboring turns, with occasional Phe residues (Figure 2b and 2c). As

120

shown in the structure-based sequence alignment for equivalent β-strands of each turn

121

(Figure 2c), PB1 has two rows of hydrophobic stacks, and PB2 and PB3 each has one

122

hydrophobic stack. Additionally, there are also polar stacks that consolidate the three-

123

dimensional structure, the well-known Asp ladder20. One ladder, consisting of seven

124

asparagine residues, is located at PB3 from turn 5 to 11, which is identical to that in 7 ACS Paragon Plus Environment

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BsIFTase. Another ladder, composed of five asparagine residues from turn 9 to 13, is

126

between PB1 and PB2 (Figure 2d). Through a symmetry operation, the overall structure of

127

AcDFA-IIIase is obtained (Figure 2e-2f), which presents a homotrimeric state and is

128

similar to that of BsIFTase.

129 130

Structure of AcDFA-IIIase in complex with DFA-III. As the crystal is grown

131

at a pH of 4.5 and temperature of 14 °C, we obtained the complex structure with

132

nondegraded DFA-III by soaking. As shown in Figure S3a, there are six

133

molecules in one asymmetric unit. Three tightly interactive molecules form a

134

regular triangle trimer (Figure 3a). The RMSD for the corresponding Cα atoms

135

between the two trimers is 0.174 Å. Thus, we only discuss one trimer below. In

136

one trimer, the RMSD of the corresponding Cα atoms between three monomers

137

is approximately 0.14 Å, suggesting that the structures of the independent

138

monomers are identical. Superimposition of unliganded and DFA-III complexed

139

form of AcDFA-IIIase shows that the RMSD is 0.138 Å, and there are rather

140

similar except some slight movements of the peripheric loops. The PB2 strands

141

of one monomer and PB3 strands in the adjacent monomer are arranged in an

142

antiparallel manner. The trimer buries 5622 Å 2 per monomer, which occupies

143

almost 30% of the surface of one monomer, as calculated by the PDBe PISA 21 .

144

There are abundant hydrogen bonds between the adjacent monomers, which are

145

important for the stabilization of the trimer. The interaction between two 8 ACS Paragon Plus Environment

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ACS Catalysis

146

adjacent monomers can mainly be divided into two parts (Figure S3b). The first

147

part is in the N-terminal region, where the long T2 loop of turn 2 is clamped by

148

the T1 loops of turns 1 and 3 of the adjacent monomer, forming 10 pairs of

149

hydrogen bonds. The second interaction is formed between the T1 loops and

150

PB2 strands of turns 4, 5, 6, 7, 8, 9 and 10 and the corresponding T2 loops and

151

PB3 strands of the adjacent molecule and contains 11 pairs of hydrogen bonds.

152 153

Substrate-binding pocket. Similar to BsIFTase, the substrate-binding pocket

154

of AcDFA-IIIase is located in the crevice between two interacting monomers 5 .

155

Structure superimposition of one monomer of each enzyme shows that the

156

RMSD for 394 C α atoms is 0.9 Å with a Dali Z-score of 58.2 (Figure S4a).

157

Therefore, there are three structurally identical and independent substrate

158

binding

159

unambiguously observed in the complex structure (Figure 3a and 3c). We named

160

the fructosyl unit F2, whose anomeric center was linked to the C-3 of the other

161

unit, which is named F1. They correspond to the F2 and F1 in BsIFTase 5 ,

162

respectively. The pocket is made up of the T1 loop of turns 4-6 of one monomer

163

and the PB3 strands and T3 loops of turns 5-7 of the adjacent monomer, forming

164

the base of the pocket, and the T2 loop of turn 2 and T3 loop of turn 9 and 11,

pockets

present

in

one

trimer.

The


substrate

DFA-III

was

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165

forming the cap of the pocket (Figure 3d). Specifically, the pocket is lined by

166

the hydrophilic residues Ser 84 , Tyr 163 , Glu 210 , Arg 258 , Glu 307 , and Gln 391 from

167

one monomer and Asp 177 , Asp 199 , and Gln 222 from the adjacent monomer, as

168

well as four hydrophobic residues, Phe 80 , Ile 85 , Phe 256, and Trp 309 . These

169

hydrophobic residues form a hydrophobic interaction with the C atom of DFA-

170

III, and the hydrophilic residues form hydrogen bonds with DFA-III directly or

171

through water (Figure 3e). Mutation of these residues to Ala reduces their

172

enzymatic activity. Particularly, mutations of residues Asp 177 , Asp 199 , Gln 222 ,

173

Arg 258 , Glu 307 , and Gln 391 completely abolished the enzymatic activity (Table

174

2).

175

Notably, the carboxyl group of Glu 210 is within 2.7 Å of the O atom in the

176

2,3’-glycosidic bond (Figure 3e). As shown in Table 2, the mutation of Glu 210

177

to Ala abolishes the activity. To explore whether the effect of the E210A

178

mutation comes from a loss of the binding ability to the substrate or the catalytic

179

ability, an ITC experiment was completed. The E210A mutation has no effect

180

on substrate binding with its K d of 50.5 ± 7.1 μM (Figure 3g). Thus, Glu 210

181

should be a catalytic residue in the enzyme. Furthermore, the E210Q mutation

182

also inactivates the enzyme and exhibits substrate binding ability with a K d of

183

14.9 ± 1.3 μM (Table 2 and Figure 3g). Collectively, these data suggest that


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ACS Catalysis

184

Glu 210 should act as the catalytic proton donor, as usually found in glycoside

185

hydrolases 22-23 . Though residue Arg 134 was far from the substrate, the R134A

186

mutation inactivates the enzyme (Table 2). Whereas Arg 134 , together with

187

Asp 199 and Gln 391 , fixes a water molecule above the anomeric center of F2 with

188

3.8 Å (Figure 3f), suggesting a possible role of this water in the hydrolysis

189

reaction.

190 191

Comparison of AcDFA-IIIase with BsIFTase. BsIFTase catalyzes the

192

degradation of inulin into DFA-III, and its structure complexed with a β-2,1-

193

linked difructosaccharide has been reported 5 . AcDFA-IIIase exhibits 50%

194

sequence identity with BsIFTase (Figure S1) and high structure homology.

195

However, in addition to some structural differences far from the substrate

196

binding pocket, a notable long insertion sequence appears to be a lid (Figure

197

S4a-S4c, Asp 378 - Asp 402 corresponding to the lid in Figure S1), covering the

198

substrate binding pocket in AcDFA-IIIase. In BsIFTase, the substrate binding

199

pocket without a lid is suitable for the long-chain substrate inulin. In contrast,

200

DFA-III is small, and in order to capture the substrate, a closed binding pocket

201

with a lid in AcDFA-IIIase seems necessary.


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202

Comparison of the substrates in the two complex structures indicates that

203

both the F1 fructosyl units are almost located at the same position, while the

204

F2 units point in different directions. With no constraints, the F2 unit in

205

BsIFTase points to the outside of the pocket, while the F2 unit in AcDFA-IIIase

206

is confined and lies at the bottom of the pocket because of the lid (Figure S4d).

207

The residues surrounding the binding pocket in the two structures are highly

208

conserved. Structure-based alignment of the residues shows that the positions

209

of the side chains are similar, except that Gln 256 in BsIFTase has an upward

210

shift to avoid clashing with the F2 unit (Figure S4d). In BsIFTase, Asp 233 looks

211

similar to a “lobster claw” to clamp the O-3 and O-4 hydroxyl groups of the F2

212

unit by forming two hydrogen bonds 5 . However, in AcDFA-IIIase, due to the

213

presence of the lid, the F2 unit of DFA-III is pushed downwards (especially by

214

Gln 391 of the lid) from Asp 199 (corresponding to Asp 233 of BsIFTase) to Asp 177

215

(corresponding to Asp 211 of BsIFTase). Consequently, Asp 177 becomes the

216

“lobster claw” that binds and orients substrates to facilitate the catalysis of

217

Glu 210 corresponding to Glu 244 in BsIFTase 5 . This binding ability is validated

218

by the ITC experiment. As shown in Figure S4e, the binding ability is abolished

219

when Asp 177 is mutated to Ala 177 (D177A), which results in enzymatic inactivity

220

(Table 2).


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ACS Catalysis

221 222

Two important residues of the lid. The Cys 387 and Gln 391 in the lid point their

223

side chains toward the substrate (Figure 4a). The side chain of Gln 391 locks

224

DFA-III in the pocket and pushes it into the proper position through a hydrogen

225

bond between the N-ε of Gln 391 and DFA-III (Figure 3e and S4d), to which the

226

catalytic residue Glu 210 can reach, which is demonstrated by the mutational

227

analysis and the ITC experiment. As shown in Table 2 and Figure 4b, the Q391A

228

mutant loses the catalytic and binding abilities for DFA-III. Specifically, the

229

O-ε of Gln 391 participates in the fixation of an important water molecule,

230

probably providing H + and OH ¯ for hydrolysis (Figure 3f), which contributes to

231

the catalytic ability. The N-ε of Gln 391 forms a hydrogen bond with DFA-III

232

(Figure 3e), which contributes to the binding ability.

233

To explore the function of Cys 387 , the mutant C387A was investigated.

234

Unexpectedly, C387A displayed a remarkable increase in catalytic activity

235

(187.51%, Table 2). To gain insights into the mechanism by which the Cys 387

236

to Ala 387 mutation increases the catalytic activity, we solved the crystal

237

structure of C387A (AcDFA-IIIase C387A ) in the unliganded-form and complex-

238

form with DFA-III. Superimposition of these structures with the native

239

unliganded-form and complex structures of AcDFA-IIIase indicates that the


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240

mutation has no influence on the overall structure (Figure 4c). Further

241

investigation of the substrate binding pockets shows that the positions of the

242

substrates and the positions of the residues encompassing the pocket between

243

the AcDFA-IIIase and AcDFA-IIIase C387A complex structures are similar

244

(Figure 4c). In addition, the distance between Cys 387 and DFA-III is 3.8 Å,

245

indicating there is no interaction between them. Therefore, the C387A mutation

246

does not affect the DFA-III binding and catalysis, which is confirmed by the

247

ITC experiment of the C387A/E210A mutant with its K d of 50.1 ± 7.2 μM

248

(Figure 4b), similar to that of E210A mutant (Figure 3g). However, the Gln 391

249

and Ala 387 residues in one monomer of the AcDFA-IIIase C387A unligadned-form

250

structure have notable movements (Figure 4c). Thus, the C387A mutation led

251

to the movement of Gln 391 . Since the side chains of Gln 391 and Cys 387

252

perpendicularly point toward the substrate, the volume of the pocket is affected

253

by Gln 391 and Cys 387 dramatically. Given that the configuration of the

254

difructosaccharide in BsIFTase is looser than DFA-III in AcDFA-IIIase (Figure

255

S4d), the C387A mutant, having a larger pocket space, should be better suited

256

to accommodate the product inulobiose, which has the same structure as

257

difructosaccharide in BsIFTase, thereby facilitating product generation.

258


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259

Decomposition of inulin by AcDFA-IIIase. Previously, it was reported that

260

DFA-IIIase from Arthrobacter sp. H65-7 has no activity towards inulin 24 . Other

261

DFA-IIIases 10-11, 13 were not studied about their activity toward inulin. However,

262

we found that AcDFA-IIIase was also able to decompose inulin in the same

263

conditions as those used for hydrolyzing DFA-III. As shown in Figure 5, after

264

incubating AcDFA-IIIase with 40 g L -1 inulin for 24 h, 54.17% of inulin (mass

265

percentage) is reserved. Simultaneously, 36.8% of DFA-III and 9.03% of

266

inulobiose are produced. Moreover, the production of DFA-III and inulobiose

267

was increased with a decreased initial concentration of inulin (Figure 5b),

268

which indicates that a high concentration of inulin has an inhibitory effect.

269

To determine the catalytic mechanism of AcDFA-IIIase for inulin, the

270

AcDFA-IIIase crystal was soaked with inulin-type saccharides that included

271

inulin, GF 2 (1-kestose), GF 3 (nystose), and GF 4 (fructofuranosyl nystose).

272

Finally, only diffraction data from AcDFA-IIIase with GF 2 were obtained

273

(Table S2). As shown in Figure 6a, there are six molecules in one asymmetric

274

unit. The RMSD for the corresponding Cα atom pairs between the two trimers

275

is 0.297 Å. Moreover, the RMSD for the corresponding Cα atom pairs among

276

the three subunits of one trimer is 0.146 - 0.154. Therefore, the overall structure

277

of each subunit is similar, and we discuss only one trimer below. For one trimer,


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

278

two GF 2 molecules are captured in the active centers, but the electron density

279

of the lid is missing. In contrast, there is one lid in the trimer unambiguously

280

observed without a GF 2 molecule captured in the active center. Superimposing

281

this structure on that of AcDFA-IIIase complexed with DFA-III, the RMSD of

282

the corresponding Cα atoms is 0.423 Å, which indicates that the overall

283

structures are the same. However, in terms of the active pocket, the glucosyl

284

unit of GF 2 conflicts with the superimposed lid from AcDFA-IIIase complexed

285

with DFA-III (Figure 6b and 6c, cyan), which probably disturbs the capture of

286

the electron density of the lid. Furthermore, the structures of AcDFA-IIIase

287

complexed with GF 2 and BsIFTase complexed with difructosaccharide were

288

superimposed (Figure S5). In terms of the active center, the residues are the

289

same. The F1 units of two substrates are well superimposed, whereas the

290

orientation of the F2 units is slightly different. In BsIFTase, the Asp 233 forms

291

bidentate hydrogen bonds with O-3 and O-4 of F2 (red dashed lines) to

292

consolidate the substrate and mediate its orientation for the nucleophilic attack

293

of Glu 244 , while this role is substituted by Arg 134 in AcDFA-IIIase

294

(corresponding to Arg 174 in BsIFTase) (black dashed lines). However, all the

295

other residues interacting with GF 2 in AcDFA-IIIase are the same with those

296

residues interacting with difructosaccharide in BsIFTase. In fact, the mutants

297

of AcDFA-IIIase E210A and E210Q lost their activities for inulin. Therefore, 16 ACS Paragon Plus Environment

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ACS Catalysis

298

AcDFA-IIIase and BsIFTase should employ the same catalytic residue (Glu 244

299

in BsIFTase and Glu 210 in AcDFA-IIIase) and catalytic mechanism for inulin.

300 301

Regulatory function of the lid. Given the two abilities of AcDFA-IIIase,

302

decomposing inulin and hydrolyzing DFA-III, the lid of AcDFA-IIIase probably

303

plays a regulatory role. To investigate this regulatory function, we constructed

304

a mutant of AcDFA-IIIase with the lid deleted (named as AcDFA-IIIase-lid¯,

305

“¯” represents “deletion”). Surprisingly, the purified AcDFA-IIIase-lid¯

306

(Figure 7a) loses the catalytic ability to DFA-III (Figure 7c), while retains the

307

ability to decompose inulin (Figure 7b and Table S1). Different substrates, GF 2 ,

308

GF 3 , and GF 4 , were also applied. The results showed that it could convert GF 3

309

to DFA-III and sucrose, GF 4 to DFA-III and GF 2 , but cannot use GF 2 as a

310

substrate (Figure 7d-7f). That is, AcDFA-IIIase-lid¯ can catalyze inulin-type

311

saccharides with more than two fructosyl residues to different products,

312

depending on the polymerization of saccharides, which is consistent with the

313

previous report 25 . Therefore, AcDFA-IIIase completely turns into an IFTase

314

(that is AcDFA-IIIase-lid¯) after removal of the lid.

315

To dissect the lid’s functions comprehensively, we solved the structure of

316

AcDFA-IIIase-lid¯. There is one molecule in one asymmetric unit of AcDFA-


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

317

IIIase-lid¯ complexed with GF 2 , with GF 2 unambiguously observed (Figure 6d).

318

When the monomers of this structure and AcDFA-IIIase are superimposed, the

319

RMSD for the corresponding C α atoms is 0.105 Å, indicating that the deletion

320

has no influence on the overall structure. Through a symmetry operation, the

321

trimeric AcDFA-IIIase-lid¯ complexed with GF 2 structure was obtained. This

322

trimeric structure is well superimposed on AcDFA-IIIase complexed with GF 2

323

(Figure 6e). The conformations of the GF 2 molecules in the pockets of the two

324

complexes are highly similar, indicating that the lid in AcDFA-IIIase

325

complexed with GF 2 is open. Furthermore, the mutation of Glu 210 to Ala 210 in

326

AcDFA-IIIase-lid¯ led to the inactivity for inulin, which indicates Glu 210 is

327

responsible for the IFTase activity of AcDFA-IIIase.

328 329

Discussion

330

Despite rigorous studies in recent years with enzymes degrading fructans to

331

DFA 8 , the mechanism of further metabolism of DFA in organisms is elusive. In

332

this work, we identified an enzyme, AcDFA-IIIase that hydrolyzes DFA-III,

333

and described its crystal structure. To our knowledge, this is the first atomic-

334

resolution structure of a DFA-hydrolase. The overall structural features of

335

AcDFA-IIIase are essentially reminiscent of a parallel β-helix. It is composed


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ACS Catalysis

336

of 13 helical turns of a right-handed coil, each of which usually contains three

337

observed β strands 18-19 . Different from the general enzymes involved in fructan

338

degradation and biosynthesis belonging to the GH32 26-27 and GH68 28-29 families,

339

DFA-IIIase belongs to the GH91 family, along with the IFTase in the CAZy

340

database 30 . Similar to BsIFTase, AcDFA-IIIase is a trimer with solid

341

interactions between adjacent monomers, and the active site is located at the

342

monomer-monomer interface. Despite the marked overall structural similarity

343

with BsIFTase, AcDFA-IIIase exhibits novel features in terms of its lid. One

344

important residue in the lid is Gln 391 , whose side chain points directly toward

345

the substrate. It forms a hydrogen bond with the O atom of the β-2’,1-glycosidic

346

bond of DFA-III and simultaneously participates in fixing a water molecule

347

(Figure 3e and 3f). Together with the functional analysis indicating that the

348

mutation of Gln 391 to Ala abolishes the binding and catalytic ability of AcDFA-

349

IIIase, we consider that Gln 391 directly pushes down and orients the substrate

350

in the correct configuration for catalysis. The lid is so critical that its deletion

351

(AcDFA-IIIase-lid¯) abolishes AcDFA-IIIase’s hydrolytic activity and turns it

352

into an IFTase.

353

Hydrolysis of the glycosidic bond has two possible mechanisms based on the

354

stereochemical configuration of the products, namely, the inversion or retention


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

355

of the anomeric configuration 31 . For DFA-III, the F1 is the β-configuration,

356

while the F2 is the α-configuration. In our complex structure, the bottom of the

357

F2 fructosyl unit of DFA-III is rather hydrophobic, composed of Phe 80 , Phe 207,

358

and Phe 256 (Figure 3e), where no water molecules exist. Furthermore, if the

359

hydrolysis product of DFA-III adopts the retaining mechanism, the initially

360

formed hydroxyl group will point at the hydrophobic core, which is not

361

beneficial. On the other hand, there is a water molecule (above F2 of DFA-III)

362

fixed by Arg 134 , Asp 199 and Gln 391 , with a distance of 3.8 Å from the anomeric

363

carbon of F2 (Figure 3f). From a structural perspective, collectively, we

364

propose that AcDFA-IIIase catalyzes the hydrolysis via an inverting mechanism

365

(Figure 8a). Generally, Asp acts as a nucleophile or a general base, and Glu

366

acts as a proton donor in the glycoside hydrolases 14 . In our structure, the

367

carboxylate group of Glu 210 is within 2.7 Å of the O atom of the α-2,3’-

368

glycosidic bond (Figure 3e). Together with the mutation of Glu 210 to Ala or Gln

369

that inactivates the enzyme, we propose that Glu 210 acts as a proton donor to

370

split the α-2,3’-glycosidic bond. This results in the generation of an O-3’

371

hydroxyl group in the F1 unit and a 2-carbonium ion in the F2 unit. Because of

372

the strong hydrophobicity of Phe 80 , Phe 207, and Phe 256 under F2, the hydroxyl

373

groups with high polarity in F2 probably are inclined to move up. There is a

374

water molecule fixed by Arg 134 , Asp 199, and Gln 391 over F2. The Asp 199 acts as 20 ACS Paragon Plus Environment

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ACS Catalysis

375

a base to deprotonate the water molecule with the formation of a hydroxide ion

376

that attacks the upward carbonium ion of F2 from the opposite side of the β-

377

2’,1-glycosidic bond. This results in the formation of the hydroxyl group at the

378

position of the anomeric carbon of F2 with a furan ring, which also inverts the

379

anomeric conformation of F2 (Figure 8a). The mutation of Asp 199 to Ala and

380

Asn leads to the inactivity of AcDFA-IIIase (Table 2), which indicates the

381

important role of Asp 199 in deprotonating the water. Furthermore, the activity

382

of the C387A mutant was substantially improved, which may be caused by the

383

shorter side chain of Ala 387 (Figure 4c) creating more space for F2 to move up

384

and the formation of the product. Moreover, it was reported that DFA-III can

385

be hydrolyzed to inulobiose as 1-O-β- D -fructofuranosyl- D -fructofuranose by

386

Arthrobacter ureafaciens 10 or as 1-O-β- D -fructofuranosyl- D -fructopyranose by

387

Arthrobacter sp. H65-7 12 . Structural and functional analyses in this study

388

demonstrate that the initial product of DFA-III is 1-O-β-D-fructofuranosyl-D-

389

fructofuranose. In solution, fructose exists as an equilibrium mixture of 70%

390

fructopyranose and 22% fructofuranose, as well as small amounts of three other

391

forms, and they all contain both an α configuration and a β configuration 32 . As

392

the anomeric center of F2 of the produced inulobiose is free, the initial product

393

1-O-β-D-fructofuranosyl-D-fructofuranose will automatically transfer to 1-O-


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

394

β-D-fructofuranosyl-D-fructopyranose until an equilibrium point is reached in

395

solution, similar to what free fructose does.

396

The structures of AcDFA-IIIase and BsIFTase are very similar except for the

397

lid. As illustrated in Figure 8b, with the lid, AcDFA-IIIase has two functions,

398

converting inulin to DFA-III and hydrolyzing DFA-III to inulobiose. That is,

399

AcDFA-IIIase is capable of sequentially catalyzing inulin, which is a novel

400

finding. As shown in Figure S1, the amino acid sequence identity between

401

AcDFA-IIIase and DFA-IIIase from Arthrobacter sp. H65-7 (AsDFA-IIIase) is

402

82% (72% for their corresponding lids). In theory, the two enzymes may have

403

the similar capability of catalyzing inulin due to this high identity. However,

404

AsDFA-IIIase shows inactivity for inulin 24 , which is probably ascribed to the

405

detection methods or reaction conditions. For the decomposition of inulin to

406

DFA-III, AcDFA-IIIase, AcDFA-IIIase-lid¯, and BsIFTase use the same

407

catalytic residues and mechanism (Figure S4d, Figure 6d and 6e, and Figure S5).

408

With this set of catalytic residues, the lid is closed when AcDFA-IIIase

409

hydrolyzes DFA-III. However, the necessary protonic states of the catalytic

410

residue Glu 210 for the decomposition of inulin and hydrolysis of DFA-III are

411

different. The states are probably regulated by the opening and closing of the

412

lid. That is, when the lid is opened substantially, Glu 210 in the general base state


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ACS Catalysis

413

is used to decompose inulin, while after binding DFA-III, it is in the general

414

acid state and can hydrolyze DFA-III. Therefore, the lid resembles a regulatory

415

switch exerting its function by its opening and closure, which is similar to the

416

mechanism of protein tyrosine phosphatase B (PtpB) 33 , whose lid serves the

417

dual requirements of recognition of substrates and protection from oxidants

418

through the switch’s on and off positions. Moreover, the lid pushes the F2 unit

419

down into the proper position, which also contributes to the hydrolysis of DFA-

420

III. These two regulatory roles, the changing protonic states of the catalytic

421

residue and the push on the F2 unit are abolished when the lid is deleted, which

422

results in the complete transformation from AcDFA-IIIase to an IFTase.

423

Although AcDFA-IIIase sequentially catalyzes inulin, it has a low catalytic

424

efficiency. As shown in Table 1, the k cat /K m of AcDFA-IIIase for inulin is 1.2

425

s -1 mM -1 . When the lid was removed (AcDFA-IIIase-lid¯ is an IFTase), k cat /K m

426

increased substantially, to 373.1 s -1 mM -1 . This is probably caused by the steric

427

hindrance of inulin’s long-chain with the lid, which decreases the affinity of

428

AcDFA-IIIase to inulin (K m for AcDFA-IIIase-lid¯ is 1.62 mM, while 18.1 mM

429

for AcDFA-IIIase). The hindrance is also shown by the structure of AcDFA-

430

IIIase in complex with GF 2 (Figure 6a-6c), in which short-chain GF 2 has already

431

perturbed the lid. On the other hand, a high concentration of inulin probably

432

occupies too many active pockets, preventing some lid closure and DFA-III 23 ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

433

hydrolysis. Therefore, lids have more opportunities to close with decreasing

434

concentrations of inulin, which increases the production of DFA-III and

435

inulobiose (Figure 5b).

436

This work reveals that DFA-IIIase and IFTase have low sequence identity

437

but highly similar three-dimensional structures, except for the lid. With this lid,

438

AcDFA-IIIase achieves sequential catalysis to inulin. Although many

439

bifunctional or multifunctional enzymes are capable of catalyzing two or more

440

reactions, they usually use different active centers or more than one catalytic

441

residue. For example, GDP-fucose synthetase 34 and GDP-mannose-3′,5′-

442

epimerase 35 adopt two sets of catalytic residues for oxidation, reduction, and

443

epimerization at the same active site. Some kinases, such as histidine kinase 36

444

and isocitrate dehydrogenase kinase 37 , phosphorylate and dephosphorylate

445

substrates at one active site using one catalytic residue. However, it is a type

446

of reverse reaction. Although vitamin K epoxide reductase converts vitamin K

447

epoxide to vitamin K and vitamin K to vitamin KH 2 using the same active site

448

and catalytic residues 38-39 , it is a type of repetitive reduction process. In

449

comparison, the interesting aspect of sequential catalysis with AcDFA-IIIase is

450

that it combines lytic reaction (IFTase activity) and hydrolytic reaction at the

451

same active site using one catalytic residue, of which the protonated states are


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ACS Catalysis

452

regulated by the lid. To the best of our knowledge, there is not another example

453

of this reaction mode that provides a similarly distinctive example of sequential

454

catalysis in enzymology. Since DFA-IIIase has IFTase function, why is IFTase

455

not substituted by DFA-IIIase in nature? As shown in Figure S1, compared with

456

DFA-IIIases, IFTases have an approximately 40~50 amino acid signal peptide.

457

Additionally, inulobiose was obtained by DFA-IIIase from Arthrobacter

458

ureafaciens ATCC21124 using only lysate and not the bacterium itself or the

459

culture broth. Therefore, IFTase and DFA-IIIase should be extracellular and

460

intracellular enzymes, respectively. IFTase is excreted extracellularly to lyse

461

large-molecule inulin, and the produced DFA-III is further hydrolyzed by

462

intracellular DFA-IIIase (Figure S6). Given the low catalytic efficiency of

463

AcDFA-IIIase for inulin with the high efficiency of AcDFA-IIIase-lid¯ (Table

464

1), the cooperation of IFTase and DFA-IIIase might be a structural evolutionary

465

relationship that produces a highly efficient utilization of inulin in nature.

466

Moreover, this work provides an example for the highly efficient production of

467

inulobiose using protein engineering. Previously, by site-directed mutagenesis

468

of the lid region, the substrate specificity, enantioselectivity, activation

469

mechanism, and stability of lipases have been significantly modified 40-42 . In

470

this work, the mutation of Cys 387 to Ala 387 in the lid increased the AcDFA-IIIase

471

activity. Therefore, the modification of the lid region deserves further 25 ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

472

exploration for the production of inulobiose with high efficiency. Additionally,

473

the production of a highly efficient synthesis of inulobiose from inulin in one

474

step using DFA-IIIase, omitting IFTase, is a challenge. Altogether, this work

475

paves the way for further metabolic investigation of inulin in nature and of

476

large-scale production, physiological function, and application of the potential

477

functional sugar inulobiose.

478 479

Methods

480

Construction, expression, and purification of enzymes. The genome

481

information of Arthrobacter chlorophenolicus A6 has been released in GenBank

482

with an accession number of CP001341.1. A putative DFA-IIIase gene

483

(GenBank ID: ACL40859.1, locus_tag: Achl_2895) is in this genome. The full-

484

length gene was commercially synthesized and ligated into the pET-22b (+)

485

vector. Based on this wild-type plasmid, mutants were constructed. All the

486

enzymes were fused with a C-terminal 6×His-tag for purification. Each plasmid

487

was transformed into E. coli BL21 (DE3). The recombinant E. coli strains were

488

inoculated into 200 mL of Luria-Bertani medium consisting of 10 g of tryptone,

489

5 g of yeast extract, 10 g of NaCl, and 1 L of distilled water. The cells were

490

cultivated at 37 °C until the OD 600 reached approximately 0.6. IPTG (1 mM of 26 ACS Paragon Plus Environment

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ACS Catalysis

491

final concentration), for the expression of enzymes, was added to the culture,

492

and the cells were cultivated for another 6 h at the same temperature. They were

493

harvested by centrifugation (12,000×g, 20 min, 4 °C), and pellets were

494

suspended in lysis buffer (50 mM sodium phosphate, 500 mM NaCl, pH 6.5).

495

After sonication (300 W; pulse on, 1 s; pulse off, 2 s; 18 min of total time) and

496

centrifugation, the supernatant was collected and loaded into the Ni 2+ -affinity

497

chromatography column pre-equilibrated with lysis buffer. The unbound

498

proteins were eluted with washing buffer (50 mM imidazole in lysis buffer),

499

while AcDFA-IIIase and its mutants were eluted with elution buffer (500 mM

500

imidazole in lysis buffer). The samples were subsequently purified with an ion-

501

exchange column (HiTrap Q Sepharose FF, GE Healthcare) and a size-exclusion

502

column (Superdex 200 10/300 GL, GE Healthcare) according to their protocols.

503

The obtained enzymes were dialyzed against buffer (50 mM sodium phosphate,

504

pH 6.5) at 4 °C overnight. The molecular weight and the purity of the enzymes

505

were determined by SDS-PAGE (stacking gel: 5%, separating gel: 12%). To

506

determine the native molecular mass of AcDFA-IIIase, gel filtration experiment

507

was performed with a column TSK G2000SWxl (Tosoh Bioscience LLC, Tokyo,

508

Japan). The mobile phase is 0.1 M phosphate buffer (pH 6.7) containing 0.05%

509

(W/V) NaN 3 and 0.1 M Na 2 SO 4 . UV was set at 280 nm on HPLC detection

510

system. The protein marker contains thyroglobulin from porcine thyroid ligand 27 ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 53

511

(66900 D a), β-amylase (200000 D a), alcohol dehydrogenase (150000 D a),

512

albumin (66000 D a), and carbonic anhydrase (29000 D a).

513 514

Identification of DFA-IIIase. The reaction solution (100 mL) contained 100 g

515

L -1 (W/V) of DFA-III, 100 nM purified AcDFA-IIIase, and 50 mM sodium

516

phosphate buffer (pH 6.5). The solution was incubated at 55 °C for 2 h, and the

517

reaction was terminated by heating the solution at 100 °C for 10 min. Thereafter,

518

the reaction solution was centrifuged (18,000×g, 4 °C, 20 min) and filtered

519

through a 0.22 μm filter membrane before loading onto a preparative column

520

(Carbomix H-NP5, 5 μm ， 10 mm id × 300 mm, Sepax, Newark, Delaware,

521

USA). The column was operated according to manufacturer protocol. The

522

fraction corresponding to the inulobiose standard (made by our previous DFA-

523

IIIase 13 ) was collected, lyophilized, and analyzed by a

524

spectrometer (Varian, Palo Alto, CA, USA).

13 C-NMR

system 300

525 526

Activity Assay. The reaction solution (1 mL) contained 10 g L -1 of substrate

527

DFA-III, 100 nM enzyme, and 50 mM sodium phosphate buffer (pH 6.5). After

528

incubation at 55 °C for 10 min, the solution was heated at 100 °C for 10 min to

529

terminate the reaction. After centrifugation (18,000×g, 4 °C, 20 min) and 28 ACS Paragon Plus Environment

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ACS Catalysis

530

filtration using a 0.22 μm filter, the filtrate was analyzed by the Sugar-Pak I

531

column (Waters, MA, USA) with an Agilent 1200 HPLC system (Agilent

532

Technologies, CA, USA) and a refractive index detector. The standard marker

533

of inulobiose was synthesized by our previously obtained DFA-IIIase 13 . The

534

amount of AcDFA-IIIase or its mutant that produces 1 μmol inulobiose per

535

minute at pH 6.5 and 55 °C was defined as one unit of activity.

536 537

Biochemical properties assay. To determine the effect of pH on enzyme

538

activity, three 50 mM buffer systems were used, including sodium citrate buffer

539

(pH 5.0 – 6.5), sodium phosphate buffer (pH 6.5 – 7.0), and Tris-HCl buffer

540

(pH 7.0 - 8.0). To determine the effect of temperature on activity, different

541

temperatures from 30 to 80 °C were used. Other conditions were the same as

542

those in the Activity assay section.

543

To determine whether AcDFA-IIIase has the ability to decompose inulin,

544

100 nM enzyme was incubated with 40 g L -1 inulin in 50 mM sodium phosphate

545

buffer (pH 6.5) (total volume is 1 mL). After incubation at 55 °C for 12 or 24

546

h, the solution was heated at 100 °C for 10 min to terminate reaction. The

547

samples were analyzed by the Sugar-Pak I column (Waters, MA, USA) with an


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

548

Agilent 1200 HPLC system (Agilent technologies, CA, USA) and a refractive

549

index detector.

550 551

Crystallization, data collection, and structure determination. The crystals

552

described in this paper were grown using the hanging-drop vapor diffusion

553

method at 287 K and grew to maximum size in approximately 1 day when mixed

554

in a 1:1 ratio with a solution containing 0.1 M sodium malonate (pH 4.2) and

555

10% PEG 3350. For data collection, crystals were transferred to cryoprotectant

556

solution consisting of the reservoir solution supplemented with 25% (v/v)

557

glycerol and then flash-cooled in liquid nitrogen. Datasets for crystals of

558

unliganded-form DFA-IIIase and its complex with substrate were collected at

559

100 K in-house, while the other crystals were collected at 100 K on the BL17U

560

synchrotron radiation beamline at Shanghai Synchrotron Radiation Facility

561

(SSRF). The datasets were processed and scaled with HKL-2000 43 and with

562

programs from the CCP4 package 44 . The structure of unliganded-form DFA-

563

IIIase was determined by molecular replacement using MOLREP 45 from the

564

CCP4 suite 44 . The structure of IFTase from Bacillus sp. Snu-7 (PDB entry:

565

2INU 5 ) was used as the search model. The initial model from MOLREP 45 was

566

refined to the full resolution range using REFMAC5 46 and manual rebuilding in


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ACS Catalysis

567

Coot 47 . The final model was evaluated with MolProbility 47 and PROCHECK 48 .

568

The other structures were determined in the same way, with the structure of

569

unliganded-form DFA-IIIase as the search model. The crystallographic

570

parameters are listed in Table S2. All of the figures showing structures were

571

prepared with PyMOL.

572 573

Isothermal titration calorimetry. The ITC binding studies were performed

574

using an ITC200 (GE) at 25 °C with 0.04 ml of 1 mM DFA-III in the injector

575

cell and 0.3 ml of 0.02 mM DFA-IIIase and the mutants in the sample cell,

576

respectively. All proteins were maintained in a buffer consisting of 25 mM Tris-

577

HCl (pH 7.5) and 200 mM NaCl. Twenty microliter injection volumes were used

578

for all experiments. Two consecutive injections were separated by 2 min to reset

579

the baseline. The control experiment, consisting of a titration of DFA-III

580

against the buffer, was performed and subtracted from each experiment. ITC

581

data were analyzed with a single-site fitting model, using Origin 8.6 (OriginLab

582

Corporation).

583 584

Functional conversion from DFA-IIIase to IFTase. Given the high similarity

585

between the AcDFA-IIIase and IFTase structures (PDB entry: 2INU or 2INV 31 ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

586

for IFTase), except for the lid (Asp 378 - Asp 402 in AcDFA-IIIase) covering the

587

substrate binding pocket, we substituted this lid and its neighbor residues

588

(Ala 376 – Met 377 and Leu 403 – A 407 , highlighted in yellow in Figure S1) with a

589

short fragment (Ser 410 to His 416 in our previous DFA-III-forming AaIFTase 13

590

sequence, Figure S1) to investigate whether AcDFA-IIIase can be converted to

591

a type of IFTase. The reaction solution consisting of 50 mM sodium phosphate

592

buffer (pH 6.5), 10 nM enzyme, and 10 g L -1 substrate inulin was used to

593

determine its IFTase activity (reaction time: 10 min, temperature: 55 °C). To

594

determine the smallest inulin-type oligosaccharide substrate of AcDFA-IIIase-

595

lid¯, a reaction solution consisting of 50 mM sodium phosphate buffer (pH 6.5),

596

10 nM enzyme, and 20 g L -1 of GF 2 , GF 3 , or GF 4 was incubated at 55 °C for 24

597

h. Other conditions and processes were referred from those of our previous

598

work 49 . To determine whether AcDFA-IIIase-lid¯ still has its original function

599

of hydrolyzing DFA-III to inulobiose, the activity was determined by the same

600

method as AcDFA-IIIase.

601 602

Data availability. Coordinates and structure factors were deposited into the

603

Protein Data Bank (PDB) under the accession codes: 5ZKS, 5ZKU, and 5ZKW

604

correspond to unliganded-form AcDFA-IIIase and its complex with DFA-III,


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ACS Catalysis

605

and 1-kestose (GF 2 ), respectively; 5ZKY and 5ZL4 correspond to the

606

unliganded-form AcDFA-IIIase-lid¯ and its complex with 1-kestose (GF 2 ),

607

respectively; 5ZL5 and 5ZLA correspond to unliganded-form AcDFA-

608

IIIase C387A and its complex with DFA-III, respectively. The data that support

609

the findings of this study are available from the corresponding authors upon

610

request

611 612

Supporting Information

613

This information is available free of charge on the ACS Publications website.

614

Multiple sequence alignment; LC-MASS spectrum; the structure of DFA-III

615

complexed form of AcDFA-IIIase; structural comparison of DFA-IIIase and

616

IFTase; the illustration of the decomposition of inulin; Table S1 and Table S2.

617 618

Additional information

619

Competing interests: The authors declare no competing interests.

620 621

Author contributions


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

622

W.M., and X.L. supervised the project. All authors were responsible for the

623

conception, design, and data analysis. S.Y., H.S., and Y.C. performed

624

experiments. S.Y., and H.S. wrote the manuscript.

625 626

Acknowledgments

627

This work was supported by the National Natural Science Foundation of China

628

Project (No. U1732114), the Postgraduate Research & Practice Innovation

629

Program of Jiangsu Province (No. KYLX16_0823), the national first-class

630

discipline program of Food Science and Technology (JUFSTR20180203)

631 632

References

633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648

1. Vijn, I.; Nevas, L.; van Kammen, A.; Franssen, H.; Bisseling, T., Nod Factors and Nodulation in Plants. Science 1993, 260, 1764-1765. 2. Roberfroid, M. B.; Van, J. A.; Gibson, G. R., The Bifidogenic Nature of Chicory Inulin and Its Hydrolysis Products. J. Nutr. 1998, 128, 11-19. 3. Van, W.; Michiels, A.; De, J.; Van, A., Fructan Biosynthetic and Breakdown Enzymes in Dicots Evolved from Different Invertases. Expression of Fructan Genes throughout Chicory Development. Sci. World J. 2002, 2, 1281-1295. 4. Avigad, G.; Bauer, S., Fructan Hydrolases. Methods Enzymol. 1966, 8, 621628. 5. Jung, W. S.; Hong, C. K.; Lee, S.; Kim, C. S.; Kim, S. J.; Kim, S. I.; Rhee, S., Structural and Functional Insights into Intramolecular Fructosyl transfer by Inulin Fructotransferase. J. Biol. Chem. 2007, 282, 8414-8423. 6. Song, K.; Bae, K.; Lee, Y.; Lee, K.; Rhee, S., Characteristics of Levan Fructotransferase from Arthrobacter ureafaciens K2032 and Difructose Anhydride IV Formation from Levan. Enzyme Microb. Technol. 2000, 27, 212218. 34 ACS Paragon Plus Environment

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649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686

ACS Catalysis

7. Cha, J.; Park, N. H.; Yang, S. J.; Lee, T. H., Molecular and Enzymatic Characterization of a Levan Fructotransferase from Microbacterium sp. AL-210. J. Biotechnol. 2001, 91, 49-61. 8. Wang, X.; Yu, S.; Zhang, T.; Jiang, B.; Mu, W., From Fructans to Difructose Dianhydrides. App.l Microbio.l Biot. 2015, 99, 175-188. 9. Haraguchi, K., Two Types of Inulin Fructotransferases. Materials 2011, 4, 1543-1547. 10. Tanaka, T.; Uchiyama, T.; Kobori, H.; Tanaka, K., Enzymic Hydrolysis of Di- D -fructofuranose-1, 2'; 2, 3'-Dianhydride with Arthrobacter ureafaciens. J. Biochem. 1975, 78, 1201-1206. 11. Neubauer, A.; Walter, M.; Buchholz, K., Formation of Inulobiose from Difructoseanhydride III Catalysed by A Lysate from Arthrobacter ureafaciens ATCC 21124. Biocatal. Biotransfor. 2000, 18, 443-455. 12. Saito, K.; Sumita, Y.; Nagasaka, Y.; Tomita, F.; Yokota, A., Molecular Cloning of the Gene Encoding the Di- D -fructofuranose 1,2 ': 2,3 ' Dianhydride Hydrolysis Enzyme (DFA IIIase) from Arthrobacter sp. H65-7. J. Biosci. Bioeng. 2003, 95, 538-540. 13. Yu, S.; Wang, X.; Zhang, T.; Stressler, T.; Fischer, L.; Jiang, B.; Mu, W., Identification of a Novel Di- D -fructofuranose 1,2’:2,3’ Dianhydride (DFA III) Hydrolysis Enzyme from Arthrobacter aurescens SK8.001. PLoS One 2015, 10, e0142640. 14. Park, J.; Kim, M. I.; Park, Y.; Shin, I.; Cha, J.; Kim, C.; Rhee, S., Structural and Functional Basis for Substrate Specificity and Catalysis of Levan Fructotransferase. J. Biol. Chem. 2012, 287, 31233-31241. 15. Herron, S. R.; Benen, J. A. E.; Scavetta, R. D.; Visser, J.; Jurnak, F., Structure and Function of Pectic Enzymes: Virulence Factors of Plant Pathogens. Proc. Natl. Acad. Sci. USA. 2000, 97, 8762-8769. 16. Larsson, A. M.; Andersson, R.; Stahlberg, J.; Kenne, L.; Jones, T. A., Dextranase from Penicillum minioluteum: Reaction Course, Crystal Structure, and Product Complex. Structure 2003, 11, 1111-1121. 17. Yoder, M. D.; Jurnak, F., Protein motifs .3. The Parallel Beta-Helix And Other Coiled Folds. FASEB J. 1995, 9, 335-342. 18. Jurnak, F.; Yoder, M. D.; Pickersgill, R.; Jenkins, J., Parallel -domains: A New Fold In Protein Structures. Curr. Opin. Struct. Biol. 1994, 4, 802-806. 19. Jenkins, J.; Pickersgill, R., The Architecture of Parallel Beta-Helices and Related Folds. Prog. Biophys. Mol. Biol. 2001, 77, 111-175. 20. Yoder, M. D.; Lietzke, S. E.; Jurnak, F., Unusual Structural Features in the Parallel Beta-Helix in Pectate Lyases. Structure 1993, 1, 241-251.


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724

21. Krissinel, E.; Henrick, K., Inference of Macromolecular Assemblies from Crystalline State. J. Mol. Biol. 2007, 372, 774-797. 22. Bujacz, A.; Jedrzejczak-Krzepkowska, M.; Bielecki, S.; Redzynia, I.; Bujacz, G., Crystal Structures of the Apo Form of Beta-Fructofuranosidase from Bifidobacterium longum and Its Complex with Fructose. FEBS J. 2011, 278, 1728-1744. 23. Ramirez-Escudero, M.; Gimeno-Perez, M.; Gonzalez, B.; Linde, D.; Merdzo, Z.; Fernandez-Lobato, M.; Sanz-Aparicio, J., Structural Analysis of Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial role of N-glycosylation in Oligomerization and Activity. J. Biol. Chem. 2016, 291, 6843-6857. 24. Sakurai, H.; Yokota, A.; Sumita, Y.; Mori, Y.; Matsui, H.; Tomita, F., Metabolism of DFA III by Arthrobacter sp. H65-7: Purification and Properties of a DFA III hydrolysis Enzyme (DFA IIIase). Biosci. Biotechnol. Biochem. 1997, 61, 989-993. 25. Uchiyama, T., Action of Arthrobacter ureafaciens Inulinase II on Several Oligofructans and Bacterial Levans. Biochem. et Biophy. Acta (BBA) Enzymology 1975, 397, 153-163. 26. Alberto, F.; Bignon, C.; Sulzenbacher, G.; Henrissat, B.; Czjzek, M., The Three-dimensional Structure of Invertase (beta-fructosidase) from Thermotoga maritima Reveals a Bimodular Arrangement and an Evolutionary Relationship between Retaining And Inverting Glycosidases. J. Biol. Chem. 2004, 279, 18903-18910. 27. Alberto, F.; Jordi, E.; Henrissat, B.; Czjzek, M., Crystal Structure of Inactivated Thermotoga maritima Invertase in Complex with the Trisaccharide Substrate Raffinose. Biochem. J. 2006, 395, 457-462. 28. Meng, G.; Futterer, K., Structural Framework of Fructosyl Transfer in Bacillus subtilis Levansucrase. Nat. Struct. Biol. 2003, 10, 935-941. 29. Martinez-Fleites, C.; Ortiz-Lombardia, M.; Pons, T.; Tarbouriech, N.; Taylor, E. J.; Arrieta, J. G.; Hernandez, L.; Davies, G. J., Crystal Structure of Levansucrase from the Gram-negative Bacterium Gluconacetobacter diazotrophicus. Biochem. J. 2005, 390, 19-27. 30. Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P. M.; Henrissat, B., The Carbohydrate-active Enzymes Database (CAZy) in 2013. Nucleic Acids Res. 2014, 42, 490-495. 31. Lammens, W.; Le Roy, K.; Schroeven, L.; Van Laere, A.; Rabijns, A.; Van den Ende, W., Structural Insights into Glycoside Hydrolase Family 32 and 68 enzymes: Functional Implications. J. Exp. Bot. 2009, 60, 727-740.


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725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762

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32. Angyal, S. J.; Bethell, G. S., Conformational Analysis in Carbohydrate Chemistry. III. The 13 C NMR Spectra of the Hexuloses. Aust. J. Chem. 1976, 29, 1249-1265. 33. Flynn, E. M.; Hanson, J. A.; Alber, T.; Yang, H., Dynamic Active-site Protection by the Tuberculosis Protein Tyrosine Phosphatase Ptpb Lid Domain. J. Am. Chem. Soc. 2010, 132, 4772-4780. 34. Somers, W. S.; Stahl, M. L.; Sullivan, F. X., GDP-fucose Synthetase from Escherichia coli: Structure of a Unique Member of the Short-Chain Dehydrogenase/Reductase Family That Catalyzes Two Distinct Reactions At the same Active Site. Structure 1998, 6, 1601-1612. 35. Major, L. L.; Wolucka, B. A.; Naismith, J. H., Structure and Function of GDP-mannose-3′ ,5′-epimerase; An Enzyme Which Performs Three Chemical Reactions at the Same Active Site. J. Am. Chem. Soc. 2005, 127 , 18309-18320. 36. Casino, P.; Rubio, V.; Marina, A., Structural Insight into Partner Specificity and Phosphoryl Transfer in Two-Component Signal Transduction. Cell 2009, 139, 325-336. 37. Zheng, J.; Jia, Z., Structure of the Bifunctional Isocitrate Dehydrogenase Kinase/Phosphatase. Nature 2010, 465, 961. 38. Chu, P.-H.; Huang, T.-Y.; Williams, J.; Stafford, D. W., Purified Vitamin K Epoxide Reductase Alone Is Sufficient for Conversion of Vitamin K Epoxide to Vitamin K and Vitamin K to Vitamin KH. Proc. Natl. Acad. Sci. USA. 2006, 103, 19308. 39. Li, W.; Schulman, S.; Dutton, R. J.; Boyd, D.; Beckwith, J.; Rapoport, T. A., Structure of A Bacterial Homologue of Vitamin K Epoxide Reductase. Nature 2010, 463, 507. 40. Shiraga, S.; Ishiguro, M.; Fukami, H.; Nakao, M.; Ueda, M., Creation of Rhizopus oryzae Lipase Having A Unique Oxyanion Hole by Combinatorial Mutagenesis in the Lid Domain. Appl. Microbiol. Biotechnol. 2005, 68, 779785. 41. Secundo, F.; Carrea, G.; Tarabiono, C.; Gatti-Lafranconi, P.; Brocca, S.; Lotti, M.; Jaeger, K.-E.; Puls, M.; Eggert, T., The Lid Is A Structural and Functional Determinant of Lipase Activity and Selectivity. J. Mol. Catal. B: Enzym. 2006, 39, 166-170. 42. Skjold-Jorgensen, J.; Vind, J.; Svendsen, A.; Bjerrum, M. J., Altering the Activation Mechanism in Thermomyces lanuginosus Lipase. Biochemistry 2014, 53, 4152-4160. 43. Otwinowski, Z.; Minor, W., Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol. 1997, 276, 307-326.


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784

44. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G. W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S., Overview of the CCP4 Suite and Current Developments. Acta Crystallogr. Section D-Biological Crystallogr. 2011, 67, 235-242. 45. Vagin, A.; Teplyakov, A., Molecular Replacement with MOLREP. Acta Crystallogr. Section D 2010, 66, 22-25. 46. Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A., REFMAC5 for the Refinement of Macromolecular Crystal Structures. Acta Crystallogr. Section D 2011, 67, 355-367. 47. Emsley, P.; Cowtan, K., Coot: Model-Building Tools for Molecular Graphics. Acta Crystallogr. Section D 2004, 60, 2126-2132. 48. Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M., PROCHECK: A Program to Check the Stereochemical Quality of Protein Structures. J. Ap. Cr. 1993, 26, 283-291. 49. Wang, X.; Yu, S. H.; Zhang, T.; Jiang, B.; Mu, W., Identification of A Recombinant Inulin Fructotransferase (Difructose Dianhydride III forming) from Arthrobacter sp 161mfsha2.1 with High Specific Activity and Remarkable Thermostability. J. Agric. Food Chem. 2015, 63, 3509-3515.


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ACS Catalysis

785

Figures

786 787 788 789 790 791 792 793 794 795 796

Figure 1. The identification and characterization of AcDFA-IIIase. (a) SDSPAGE of wild-type AcDFA-IIIase and its mutants. (b) HPLC profile of reaction mixture (AcDFA-IIIase converts DFA-III to inulobiose) after 24 h, which was detected by by Sugar-Pak I column (Waters, MA, USA). (c) 13 C-NMR spectrum of inulobiose in b. The specific peaks at 98.12 and 101.58 ppm can be used to distinguish the C-2 of reducing fructose in pyranose and furanose. The detailed values are summarized in Table S1. (d) The effect of pH on AcDFA-IIIase activity. (e) The effect of temperature on AcDFA-IIIase activity. (f) Determination of native molecular mass of AcDFA-IIIase using gel filtration. The protein marker contains thyroglobulin from porcine thyroid ligand (66900 39 ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

797 798 799

D a), β-amylase (200000 D a), alcohol dehydrogenase (150000 D a), albumin (66000 D a), and carbonic anhydrase (29000 D a). The retention time of AcDFA-

IIIase is 8.6 min.


Page 40 of 53

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ACS Catalysis

800 801 802 803 804 805

Figure 2. The crystal structure of unliganded-form AcDFA-IIIase monomer. (a) Side view of monomeric AcDFA-IIIase. PB1, PB2, and PB3 β-strands are colored green, blue, and red, respectively. From turn 1 to 13, they form PB1, PB2, and PB3 β-sheets, respectively. Two long loops form the active center, T3 from turn 11 and T2 from turn 2, and are labeled T3 and T2, respectively. The 41 ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

806 807 808 809 810 811 812 813 814 815

N- and C-termini are labeled. (b) The side chains of internal stacked hydrophobic residues are presented with sticks in a view from the C-terminus. (c) Structure-based sequence alignment for β-strands in PB1, PB2, and PB3 βsheets. Colors are identical to those in (b). The internal stacked hydrophobic residues in (b) are boxed. (d) Asparagine ladders with red dash circles are formed by stacked asparagine residues. One ladder consisting of 7 asparagine residues is located at PB3 β-strands from turn 5 to 11. Another one composed of five asparagine residues from turn 9 to 13 is between PB1 and PB2. (e) Top and bottom view of trimeric unliganded-form AcDFA-IIIase structure. (f) Electrostatic surface of the trimer in (e), which calculated by Pymol software.

816


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43 817

ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833

Figure 3. The structure of AcDFA-IIIase in complex with DFA-III. (a) AcDFA-IIIase is a homotrimer. Three DFA-III molecules presented with sticks (cyan) are located at the monomer-monomer interface. (b) The structural comparison of the trimeric unliganded-form (cyan) AcDFA-IIIase and DFA-III complexed form AcDFA-IIIase (pink). (c) The 2Fo-Fc electron density map contoured at 1.0 σ is overlaid on the model of DFA-III in the substrate binding pocket. The fructosyl units are labeled with F1 and F2 as those of difructosaccharide are labeled in BsIFTase 5 . The numbers 1 and 2 represent C-1 and C-2 atoms of F2, while primed numbers refer to the C-2 and C-3 atoms of F1. (d) DFA-III presented with sticks (cyan) in the active pocket is located at the monomer-monomer interface. (e) The interactions between DFA-III and residues of AcDFA-IIIase. The red dashed lines are hydrogen bonds. The carboxyl group of Glu 210 is within 2.7 Å of the O atom of the 2,3’-glycosidic bond. (f) A water molecule above F2 probably plays a critical role in the hydrolysis of DFA-III. (g) Isothermal titration calorimetric analysis of DFA-III into E210A and E210Q.

834


Page 44 of 53

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ACS Catalysis

835 836 837 838 839 840 841 842 843 844 845 846 847

Figure 4. The crystal structure of mutant C387A. (a) The molecular surface of the substrate-binding site is shown with DFA-III, Gln 391 , and Cys 387 represented by a stick model. The lid drawn with slight transparency is colored with light blue. (b) Isothermal titration calorimetric analysis of DFA-III into mutant Q391A and C387A/E210A. (c) Structural superimposition of AcDFAIIIase (unliganded-form is colored with blue, complex-form with DFA-III is colored with pink), AcDFA-IIIase C387A (unliganded-form is colored with green, complex-form with DFA-III was colored with cyan). The conformations and positions of substrate DFA-III are the same. The residues in active centers are well superimposed except Gln 391 in chain A of the unliganded-form of AcDFAIIIase C387A (in the red circles). The conformation of this Gln 391 is substantially changed, and the position of Ala 387 is changed.


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

848 849 850 851 852 853 854 855 856 857

Figure 5. Decomposing inulin by AcDFA-IIIase. a. HPLC profiles of mixtures after AcDFA-IIIase catalyzes inulin. The residual inulin, produced DFA-III, produced inulobiose were labeled on the profile. At 12 h, there were no other sugars produced, while other sugars as by-products were produced at 24 h. b. AcDFA-IIIase catalyzes inulin with different concentrations of inulin with the same conditions in a. The mass percentages represent the mass ratios of residual inulin, produced DFA-III, and produced inulobiose, which are used to indicate how the concentrations of initial inulin influence the production of DFA-III and inulobiose. The calculation formula is, for example, the mass percentage of residual inulin = W inulin /(W inulin + W produced DFA-III + W produced inulobiose ). Here, W represents mass. We excluded other sugar of by-products in the formula because it is hard to qualify and quantify them due to the little amount. The experiments were performed with three replications.


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858 859

Figure 6. The structure of AcDFA-IIIase complexed with GF 2 (a-c) and

860

AcDFA-IIIase-lid¯ (d-e). (a) Six molecules (two trimers, gray) in one

861

asymmetric unit of AcDFA-IIIase complexed with GF2. The lid is colored pink. 47 ACS Paragon Plus Environment

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

862

For one trimer, two GF 2 molecules were captured, but the electron density of

863

the lids on top of the GF 2 molecules was lacking, so these lids were not included

864

in the final model (red circles). In contrast, the lid with the α-helix was

865

unambiguously observed in the pocket without GF 2 . The structure of GF 2 was

866

shown and G represents the glucosyl unit. (b - c) The superimposition of

867

AcDFA-IIIase complexed with DFA-III (cyan) on its complex with GF 2 (gray).

868

The RMSD between the Cα atoms of the two structures is 0.423. DFA-III

869

(labeled with red color) and the GF 2 molecule (labeled with black color) are

870

presented as sticks. The glucosyl unit of the GF 2 molecule conflicts with the lid

871

(α-helix) from AcDFA-IIIase complexed with DFA-III (cyan), which leads to

872

the lack of electron density of the lid and only part of the lid (pink) was captured.

873

(d) The superimposition of AcDFA-IIIase-lid¯ complexed with GF 2 (blue) on

874

AcDFA-IIIase complexed with DFA-III (cyan). The fructosyl units F1 and F2

875

correspond to those of DFA-III in AcDFA-IIIase and difructosaccharide in

876

BsIFTase 5 . G represents the glucosyl unit. (e) The superimposition of AcDFA-

877

IIIase-lid¯ complexed with GF 2 (blue) on AcDFA-IIIase complexed with GF 2

878

(gray). GF 2 molecules between the two structures are well superimposed.


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879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894

ACS Catalysis

Figure 7. The identification and characterization of AcDFA-IIIase-lid¯. (a) SDS-PAGE of AcDFA-IIIase-lid¯. (b) HPLC profile of reaction mixture (AcDFA-IIIase-lid¯ with substrate inulin) after 24 h. The produced DFA-III was further identified by 13 C-NMR spectrum (the inset). The detailed values of chemical shifts of carbons are summarized in Table S1. (c) HPLC profile of reaction mixture (AcDFA-IIIase-lid¯ with DFA III) after 12 h, which was detected by Sugar-Pak I column (Waters, MA, USA). Nothing was produced. (d) - (f) HPLC profiles of reaction mixture (AcDFA-IIIase-lid¯ with GF 2 , GF 3 , and GF 4 , respectively) after 48 h. GF 2 , GF 3 , and GF 4 are 1-kestose, nystose, and fructofuranosyl nystose, respectively. They are used to identify the smallest substrate of AcDFA-IIIase-lid¯. The smallest substrate is defined as an inulin-type oligosaccharide substrate with the shortest chain length. The profiles of b, d – f were detected using Asahipak NH2P-5004E column (4.6 mm × 250 mm, shodex, Tokyo, Japan) referring to previous work 49 , while profile in c was with Sugar-Pak I column.

895


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

896 897 898 899 900 901 902 903 904

Figure 8. Proposed catalytic and regulatory mechanism of AcDFA-IIIase. (a) Details of the mechanism are described in the text. The black dot labeled with W represents a water molecule. The primed residues (D199 and R134) are located at the adjacent subunit. (b) The reaction of AcDFA-IIIase with inulin and DFA-III. The lid is open when AcDFA-IIIase reacts with long-chain inulin, while it is closed when it reacts with short-chain DFA-III. The lid of AcDFAIIIase is colored red. The dashed line represents inulobiose and is produced by further hydrolyzing DFA-III using AcDFA-IIIase itself.

905


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ACS Catalysis

906

Tables

907

Table 1. The Kinetic Parameters. All of the reactions were at pH of 6.5 (sodium phosphate buffer) and 55 °C. 100

908

nM AcDFA-IIIase and AcDFA-IIIase C387A were used to react with 2.0 ~ 50 mM DFA-III for 10 min while 24 h for 0.5

909

~ 20 mM inulin. 100 nM AcDFA-IIIase-lid¯ was used to react with 0.5 ~ 20 mM inulin for 10 min. The experiments

910

were perfomed with three replications. K m (mM)

k cat /K m (s -1 mM -1 )

121.30 ± 12.8

4.61 ± 0.16

AcDFA-IIIase C387A for DFA-III

98.06 ± 6.9

8.59 ± 0.42

AcDFA-IIIase for inulin

18.1 ± 0.9

1.2 ± 0.09

AcDFA-IIIase-lid¯ for inulin

1.62 ± 0.04

373.10 ± 19.57

Samples AcDFA-IIIase for DFA-III

911


ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

912

Page 52 of 53

Table 2. The Activity of AcDFA-IIIase and Its Mutants. Samples

Specific activity (U mg -1 )

Relative activity (%)

AcDFA-IIIase

101.25 ± 6.04

100.00

C387A

187.51 ± 5.14

185.20

C387A/E210A

0.00

0.00

D177A

0.00

0.00

D177N

0.00

0.00

D199A

0.00

0.00

D199N

0.00

0.00

E210A

0.00

0.00

E210Q

0.00

0.00

E307A

0.00

0.00

F80A

8.71 ± 1.02

8.61

F207A

67.72 ± 2.45

67.72

F256A

17.87 ± 0.98

17.65

I85A

14.93 ± 1.66

14.75

Q222A

0.00

0.00

Q391A

0.00

0.00

R134A

0.00

0.00

R258A

0.00

0.00

S84A

10.45 ±1.01

10.32

W309F

5.17 ± 0.49

5.10

Y163A

8.19 ± 0.91

8.09

913

a

914

acid was the average value of three replications.

a

The relative activity of AcDFA-IIIase is defined as 100%, The data of specific


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915

ACS Catalysis

Table of contents graphic

916 917


Structural and Functional Basis of Difructose Anhydride III Hydrolase

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