Computation-Aided Rational Deletion of C-Terminal Region Improved

Subscriber access provided by Kaohsiung Medical University

Biotechnology and Biological Transformations

Computation-aided rational deletion of C-terminal region improved the stability, activity and expression level of GH2 #-glucuronidase Beijia Han, Yuhui Hou, Tian Jiang, Bo Lv, Lina Zhao, Xudong Feng, and Chun Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03449 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

Journal of Agricultural and Food Chemistry

1

Computation-aided rational deletion of C-terminal region improved

2

the stability, activity and expression level of GH2 β-glucuronidase

3 4 5

Beijia Han†,⊥, Yuhui Hou†,⊥, Tian Jiang†, Bo Lv†, Lina Zhao§, Xudong Feng*,†, Chun Li*,†

6 7

†Institute

8

and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China.

9

§Key

for Synthetic Biosystem/Department of Biochemical Engineering, School of Chemistry

Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High

10

Energy Physics, Chinese Academy of Sciences, Beijing 100049, China.

11

*Correspondence

12

⊥These authors

to Xudong Feng ([email protected]) and Chun Li ([email protected])

contributed equally to this work.

1

ACS Paragon Plus Environment


14 15

ABSTRACT

16

In this study, computation-aided design on the basis of structural analysis was employed to

17

rationally identify a highly dynamic C-terminal region that regulates the stability, expression level

18

and activity of a GH2 fungal glucuronidase from Aspergillus oryzae Li-3 (PGUS). Then, four

19

mutants with a precisely truncated C-terminal region in different lengths were constructed, among

20

them, mutant D591-604 with a 3.8-fold increase in half-life at 65 °C and a 6.8 kJ/mol increase in

21

Gibbs free energy showed obviously improved kinetic and thermodynamic stability compared to

22

PGUS. Mutants D590-604 and D591-604 both showed approximately 2.4-fold increases in the

23

catalytic efficiency kcat/Km and 1.8-fold increases in the expression level. Additionally, the

24

expression level of PGUS was doubled through a C-terminal region swap with bacterial GUS from

25

E. coli (EGUS). Finally, the robust PGUS mutants D590-604 and D591-604 were applied in the

26

preparation of glycyrrhetinic acid with a 4.0- and 4.4-fold increases in concentration through

27

glycyrrhizin hydrolysis by a fed-batch process.

28

Keywords: β-glucuronidase; molecular dynamics simulation; protein engineering; C-terminal

29

region; glycyrrhetinic acid

30

2


Page 2 of 39

Page 3 of 39


31

INTRODUCTION

32

Glucuronide conjugates are an important class of natural product compounds, in which the

33

bioactive aglycone is decorated with glucuronic acids. Glucuronides can be derived by

34

glucuronidation based on various carbon frameworks such as terpenoids, flavones and alkaloids,

35

which were widely applied in the food, cosmetic and pharmaceutical industry.1-3 The glucuronic

36

acid moieties play an important role in regulating the solubility, function and bioavailability of the

37

glucuronides, but overdecoration with glucuronic acid may also introduce negative effects.4-6

38

Traditionally, the excess sugar is removed by acid/base hydrolysis, but this process needs a high

39

pressure and high temperature, and the reaction is uncontrollable. A more efficient and green

40

process for the transformation of glucuronides is highly desirable. β-Glucuronidases (GUSs, EC

41

3.2.1.31), as an important class of glycoside hydrolases (GHs), cleave the β-glucuronidic bond

42

from the nonreducing end and release the glucuronic acid moieties.7 Most GUSs identified so far

43

belong to GH2, and the crystal structures of GUS from humans (HGUS), Escherichia coli (EGUS),

44

and Firmicutes in GH2 have been resolved.8-10 Recently, GUSs have drawn special attention in

45

modifying natural glucuronides due to the mild reaction conditions and controllable process.5, 6, 11,

46

12

47

the complexed components in the natural products may also inhibit the activity of GUSs. Therefore,

48

increasing the stability and activity of GUSs under harsh reaction conditions has become an

49

important issue, as it will assist the enzyme in resisting the deactivation of the intricate substrate

50

and allow elevating the reaction temperature to increase the reaction efficiency.13, 14

51

However, the poor solubility of the natural products has limited the hydrolysis efficiency, and

Directed evolution is the most widely used way to improve enzyme stability, but this method 3



Page 4 of 39

52

is labor-intensive and requires high-throughput screening.15-18 With the development of structural

53

biology, many relationships between the enzyme structure and the stability/other functions have

54

been revealed. The mutagenesis of a single or several key residues based on structural analysis or

55

sequence alignment has been reported to improve the enzyme stability, which significantly

56

simplifies the experimental task.19-22 Recently, loop engineering has drawn special attention to

57

improving the enzyme stability due to the exposure to the solvent and the likeliness of establishing

58

interactions with the substrate.23, 24 Flexible surface loops have been considered “hot regions” for

59

engineering, therefore, loop truncation

60

significantly improve the enzyme stability. However, it is more important to precisely target these

61

loops for mutagenesis due to the associated large reorganization of the tertiary structure. Although

62

several strategies have been proposed based on the B-factor or sequence alignment, more efforts

63

are still required to achieve rational design for identifying unstable loops. Recently, computational

64

modeling methods such as molecular dynamics (MD) simulations have also been applied to assist

65

in mutation design,30-33 providing ideas and a theoretical basis to guide experiments. Therefore, it

66

is anticipated that the combination of structural analysis and computation may be a promising

67

pathway for the efficient design of mutants.

25-27

and replacement

28, 29

have been reported to

68

Glycyrrhizin (GL), the main compound in licorice, has anti-inflammatory, anti-allergic, and

69

anti-cancer activities, but its strong polarity decreases its bioavailability.34-37 GL can be

70

transformed into glycyrrhetinic acid (GA) by removing two glucuronic acid moieties. GA has

71

better absorption efficiency and all the functions of GL, therefore, was considered an important

72

pharmaceutical ingredient.37, 38 In our group, a β-glucuronidase was identified from Aspergillus 4


Page 5 of 39


73

oryzae Li-3 and heterologously expressed in Escherichia coli (PGUS), where it hydrolyzed GL

74

into GA with high efficiency.4 However, the poor stability and low activity of PGUS greatly

75

increased the reaction cost, severely limiting its industrial application. Recently, we resolved the

76

crystal structure of PGUS (PDB ID: 5C70), which represents the first structure of a fungal GUS.4

77

In this research, the stability, expression level and activity of PGUS were simultaneously improved

78

by rationally deleting its C-terminal region, which was a surface dynamic region identified by

79

structural analysis and MD simulation, that regulated the function of PGUS. The C-terminal region

80

deletion of PGUS enhanced not only enzyme stability but also the expression level and catalytic

81

efficiency. By investigating the functional roles of the C-terminal regions of other GH2 GUSs, we

82

found that this region was an evolutionary feature of GH2 GUSs. Our work not only presents a

83

robust industrial biocatalyst for the modification of natural glucuronide compounds but also

84

illustrates the implication of the C-terminal region in GH2 GUSs evolution.

85

MATERIALS AND METHODS

86

Materials

87

The pgus (GenBank, EU095019.1) gene encoding β-glucuronidase from Aspergillus oryzae Li-3

88

was saved in our lab.4 The egus gene was directly cloned from E. coli BL21(DE3) strain, and the

89

hgus gene was synthesized by Hongxun (Beijing, China) based on the deposited protein sequence

90

(GenBank, AAA52561.1). E. coli TOP10 and BL21(DE3) competent cells were purchased from

91

Biomed (Beijing, China). The DNA polymerase, restriction enzymes and T4 ligase were purchased

92

from TransGene Biotech (Beijing, China). GL and 4-nitrophenyl-β-D-glucuronide (pNPG) were 5



93

purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium glycyrrhizinate was purchased

94

from Xinjiang Tianshan Pharmaceutical Co. (China). All other chemicals were of analytical grade.

95

Bioinformatic analysis

96

The values of the B-factor and root mean square fluctuation (RMSF) were used to evaluate the

97

protein flexibility, and the high value represented high flexibility. The B-factor was calculated

98

with B-FITTER39 software from the PGUS crystal structure, and the RMSF was calculated with

99

VMD (Visual Molecular Dynamics) software from the MD simulation.40 The loops with high B-

100

factors and RMSF values were considered unstable regions of PGUS. The PGUS mutants with

101

deleted unstable loops were computationally constructed with VMD software, and the structures

102

of PGUS and mutants were displayed and analyzed by PyMOL2.7.

103

MD simulation

104

In the crystal structure of dimeric PGUS (PDB ID: 5C70), several regions were missed due to the

105

poor electron density. Therefore, these missed regions were first reconstructed by molecular

106

modeling. The missed residues of loop 25-29, loop 364-372 and loop 593-604 were added to the

107

PGUS structure by using the built-in psfgen package of VMD software combined with the

108

top_all27_prot_lipid (the topology file). Then, the conformation optimization of the reconstructed

109

enzyme was facilitated by relaxing only the added amino acids until the complete system became

110

stable. Based on this structure, mutants with deleted C-terminal regions were generated by

111

removing relevant amino acids, and the missed H or OH at the end of the broken peptide bond was

112

added. The obtained structure was used as starting point for the all-atom MD simulation.

113

Each system was solvated using TIP3P41 with the water box extended to 15 Å. Periodic 6


Page 6 of 39

Page 7 of 39


114

boundary conditions were applied for energy minimization and equilibration, and the solvated

115

protein was ionized using 0.15 M NaCl to neutralize the charge. All MD simulations were

116

performed with NAMD 2.9. The initial speed of all atoms was set to zero, and then, their relative

117

locations were changed instantly to identify the lowest energy spot, which was used as the MD

118

simulation original state. After minimization, the temperature was increased from 0 K to 310 K in

119

the NVT, and long-range electrostatic effects were modeled using the partial-mesh Ewald

120

method.42 The switching function was employed to guarantee that the van der Waals and

121

electrostatic forces could transit to zero smoothly at the cutoff point. All bonds with hydrogen

122

atoms were fixed to decrease the computation task. The trajectory was recorded every 5000 steps

123

(1 step being 2 fs), and the par_all27_prot_lipid parameter was applied to all calculations in the

124

CHARMM force field.

125

Construction of GUS mutants

126

The three different GUS genes (pgus, egus and hgus) were added into pET-28a (+) independently

127

and used as the template for mutagenesis. Briefly, the forward primer containing the EcoR I site

128

and the reverse primer (matching the desired mutants) containing the Not I site were designed and

129

used to amplify the GUS genes (the primers are listed in Table S1). The scheme of the plasmid

130

construction is shown in Fig. S1. The PCR products were ligated by T4 DNA ligase and

131

transformed to E. coli TOP10 competent cells to enrich the plasmid. The resulting plasmids were

132

transferred to E. coli BL21(DE3) competent cells for protein expression. The gene sequencing was

133

performed by Genewiz (Beijing, China).

7



134

Enzyme expression and purification

135

The seed solution of GUSs wild-type and mutants were incubated overnight in Luria-Bertani

136

medium containing 50 μg/mL kanamycin sulfate at 37 °C with shaking. Then, the seed was

137

inoculated into a fresh Luria-Bertani medium at a 1% volume fraction and cultured under the same

138

conditions. When OD600 reached approximately 0.5-0.6, the protein expression was induced by

139

adding 1 mM isopropyl β-D-thiogalactoside (IPTG), followed by incubation at 16 °C for 14 h. The

140

cells were collected by centrifugation at 6000 rpm for 10 min and then resuspended in buffer A

141

(50mM Tris-HCl, pH 7.4, 150 mM NaCl). Then, the cells were lysed by a high-pressure crushing

142

apparatus at 4 °C, and after centrifugation at 12000 rpm for 20 min, the supernatant was reserved

143

as a crude enzyme. The target enzyme was eluted with 15% buffer B (50 mM Tris-HCl, pH 7.4, 1

144

M imidazole) and desalted with a PD-10 desalting column with 50 mM Tris-HCl (pH 7.3) (GE

145

Healthcare, Beijing, China) as an elution buffer. All enzyme purification was conducted in an

146

ÄKTA purifier (GE Healthcare, Beijing, China), and the enzyme molecular weight, expression

147

level and purity were analyzed by SDS-PAGE. The protein concentration was assayed at 595 nm

148

by the Bradford method according to the standard of bovine serum albumin.43

149

Enzyme activity assay and kinetics characterization

150

The activity of PGUS, EGUS and the mutants was determined by pNPG hydrolysis. The reaction

151

was initiated by adding 20 μL purified enzyme into 30 μL 0.79 mM pNPG (pH 5, 50 mM acetate

152

buffer), followed by incubation at 40 °C for 5 min. The reaction was halted by adding 150 μL 0.4

153

M NaCO3, and the OD405 of the resulting sample was measured by ELx808 (BioTek, Beijing,

154

China) to evaluate the produced pNP. One enzyme unit was defined as the amount of enzyme 8


Page 8 of 39

Page 9 of 39


155

required to produce 1 μmol 4-nitrophenol in one minute under the above reaction conditions. The

156

Michaelis−Menten kinetics were also evaluated by pNPG hydrolysis, with the concentration

157

ranging from 0.12 to 2.76 mM. The kinetic parameters were calculated by nonlinear fitting with

158

the curve fitting program of MATLAB.

159

Stability determination

160

The kinetic thermostability of PGUS, EGUS and the mutants was determined by incubation at

161

70 °C for 100 min. Samples were taken every 20 min and put in an ice bath immediately for 20

162

min to recover the reverse deactivation. The residual activity was calculated by the pNPG assay as

163

described above. The activity of the unincubated sample was taken as 100%.

164

The thermodynamic stability of PGUS and the mutants was determined by guanidine

165

hydrochloride (GdnHCl)-induced denaturation. The purified enzyme (0.2 mg/mL) was incubated

166

in 0.12 M-4.4 M GdnHCl solution containing 10 mM NaCl and 10 mM CaCl2, and the mixture

167

was incubated at 25 °C for 2 h. The intrinsic fluorescence of the denaturation process was

168

monitored at an excitation wavelength of 280 nm in a 300-mm quartz cell using the fluorescence

169

spectrophotometer FluoroMax-4 (HORIBA, USA), and the fluorescence intensity at an emission

170

wavelength of 330 nm was recorded for further calculation. ΔG, the Gibbs free energy of unfolding

171

in water, was calculated as described by Nick Pace et al.44, 45 In addition, the fluorescence spectrum

172

of each enzyme without the addition of GdnHCl was scanned to determine the tertiary structure

173

changes caused by mutation.

174

Circular dichroism (CD) analysis

175

The CD spectra was measured with a J-810 CD spectropolarimeter (JASCO, Tokyo, Japan) to 9



176

investigate the effect of mutation on the enzyme secondary structure. All PGUS enzyme samples

177

(0.2-0.3 mg/mL) were prepared in 20 mM NaAC-HAC buffer (pH 4.5) and placed into a quartz

178

cell with a 0.1-cm slit. The CD spectra were recorded with far-ultraviolet wavelengths ranging

179

from 190 nm to 240 nm, and the CD fitting curve and the content distribution of secondary

180

structure were analyzed by jwexpl 32 software.

181

Biotransformation of glycyrrhizin

182

PGUS, D590-604 and D591-604 were selected for a further GL hydrolysis assay to investigate

183

their performance in practical applications. The reactants, consisting of 30 mL crude enzymes and

184

70 mL GL (2 g/L ammonium glycyrrhizinate, dissolved in 50 mM acetate buffer, pH 4.5), were

185

incubated at 40 °C, and fresh GL was fed to the reactant vessels within a certain interval. Samples

186

(97 μL) were taken at certain time and mixed with 3 μL NaOH to stop the reaction, and the

187

substrate and product were analyzed by HPLC. The reactant (100 μL) was mixed with 900 μL

188

methanol, and the mixture (10 μL) was injected into a C18 column (4.6 × 250 mm, 5-μm particle

189

size, Shimadzu). The separation was achieved with a mobile phase consisting of methanol and 6 ‰

190

acetic acid solution (84:16 v/v), and the eluate was monitored with an UV detector at 254 nm. The

191

GL conversion (CGL) was calculated by CGL(%)=(S0-St)/St×100, where S0 stands for the total GL

192

concentrations provided and St stands for the GL concentration at time t. The GA yield (YGA) was

193

calculated by YGA(%)=SGA/(SGA+SGAMG) × 100, where SGA and SGAMG stand for the molar

194

concentrations of GA and glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG), respectively.

195

RESULTS

196

Discovery of dynamic region by structural analysis and MD simulation 10


Page 10 of 39

Page 11 of 39


197

The B-factor is a critical factor to evaluate the flexibility of an individual residue in a protein.46

198

However, in our case, the B-factors of several regions of PGUS including loop 25-29, loop 364-

199

372 and loop 593-604 were not available owing to the poor electron density,4 therefore these

200

missed regions were first reconstructed by MD simulation. RMSF reflects the stability of each

201

residue over the MD simulation process. Therefore, a combination of B-factor and RMSF analysis

202

was used to target the unstable regions of PGUS. As shown in Fig. 1, RMSF generally corresponds

203

well to the B-factor value. Notably, the available part of the C-terminal region from the crystal

204

structure showed remarkably high B-factor values with N591 of 83.33 and L592 of 90.01, while

205

the rest of the reconstructed C-terminal region also showed a high RMSF, with average value

206

above 12 Å. This value is much higher than that of other regions, indicating that the C-terminal

207

region is highly flexible, which may be responsible for the instability of PGUS. The reconstructed

208

conformation of the C-terminal region is shown in Fig. 1c. This region formed part of the catalytic

209

TIM-barrel domain but was located far from the substrate channel and active center of PGUS. In

210

addition, this region was in a random coil conformation and had no interaction with neighboring

211

residues. Theoretically, the deletion of the C-terminal region would not have negative effect on

212

the catalytic activity. In contrast, the N-terminal region (residues 1-9) of PGUS was quite stable,

213

which was significantly different from the case in enzymes in other GH families, including

214

glucanase in GH547 and xylanase in GH11 and GH10,28, 48, 49 where the N-terminal region has been

215

reported to play critical roles in enzyme stability. Therefore, on the basis of the above rational

216

analysis, the deletion of the C-terminal region with various lengths was performed to enhance the

217

PGUS stability, yielding four mutants: D590-604, D591-604, D595-604 and D597-604 (the 11



218

mutants were named DX-X, where X-X stands for the deleted residues). In addition, to compare

219

with other GHs, a PGUS mutant with the deleted N-terminal region was also constructed: D1-9.

220

To reduce the wet-experiment task, the above five mutants were first constructed by

221

computation modeling to test whether the mutagenesis could enhance the enzyme stability via a

222

95-ns MD simulation. As shown in Fig. 2a, mutants D590-604 and D591-604 had lower root mean

223

square deviation (RMSD) values than PGUS, indicating that a more stable structure was obtained.

224

Mutant D597-604 showed a lower RMSD than the wild-type before 75 ns, but the values then

225

became similar. Mutant D595-604 showed a similar RMSD value to PGUS over the entire MD

226

simulation. As expected, mutant D1-9 showed an even higher RMSD value than PGUS, indicating

227

that the deletion of the N-terminal region has a detrimental effect on the enzyme stability (Fig. 2b).

228

The positive mutants with a truncated C-terminal region were subjected to experimental tests.

229

Deletion of the C-terminal region improved the robustness of PGUS

230

PGUS and its mutants were experimentally constructed and purified to a purity higher than 90%

231

(Fig. S2), and their kinetic thermostability was investigated at 65 °C and 70 °C. As shown in Fig.

232

3a, mutants D590-604 and D591-604 showed dramatically improved thermostability at 65 °C

233

compared to PGUS. The half-life (t1/2) of D591-604 was estimated to be 693 min based on first-

234

order deactivation kinetics, which is 511 min longer than that of PGUS (182 min). D597-604

235

showed similar thermostability to PGUS, while D595-604 showed lower thermostability. D590-

236

604 and D591-604 still displayed much better performance even at 70 °C, for example, D590-604

237

and D591-604 still retained 80% activity after 100 min incubation, while PGUS had only 16%

238

activity left. The t1/2 of D591-604 (117 min) was 70 min longer than that of PGUS (47 min) at 12


Page 12 of 39

Page 13 of 39


239

70 °C. Incubation for 100 min at 70 °C seemed to be the turning point, since mutants D590-604

240

and D591-604 showed severe activity losses after 100 min, with the residual activity decreasing

241

from 80% to 34% and 45% in 20 min, respectively. D597-604 showed slightly better

242

thermostability than PGUS at the first stage, but it was quickly deactivated after passing the turning

243

point. It seems that the thermostability improved with the increasing number of residues deleted

244

from the C-terminal region. The thermostability trend is in agreement with the RMSD results in

245

Fig. 3, indicating that the computation is reliable for the rational design of mutants.

246

The thermodynamic stability of PGUS and the mutants were investigated by monitoring the

247

intrinsic fluorescence variation during guanidine hydrochloride (GdnHCl)-induced denaturation.

248

As shown in Fig. 4, the Gibbs free energies of unfolding (ΔG) of D590-604 and D591-604 were

249

3.0 kJ/mol and 6.8 kJ/mol higher than that of PGUS (31.9 kJ/mol), respectively, indicating that

250

both of the mutants had more stable structures. D595-604 showed a similar ΔG as PGUS (31.6

251

kJ/mol), while D597-604 showed a 10.3 kJ/mol lower ΔG. In summary, mutants D590-604 and

252

D591-604, with the deletion of the whole C-terminal region, showed dramatically improved

253

kinetic and thermodynamic stability compared to the wild-type enzyme.

254

The kinetics of PGUS and the mutants were characterized by pNPG hydrolysis. As shown in

255

Table 1, the Km values of D591-604 and D595-604 were similar to that of PGUS, while the Km

256

values of D590-604 and D597-604 decreased by 58% and 50%, respectively, indicating that these

257

mutants had enhanced binding affinity. In addition, mutant D590-604 showed a similar kcat to

258

PGUS, while all other mutants showed 19-163% increases. In summary, all mutants showed 65-

259

143% increases in the catalytic efficiency kcat/Km over that of PGUS. These results indicate that 13



260

mutants D590-604 and D591-604 exhibit not only improved stability but also increased catalytic

261

efficiency kcat/Km (increasing by 2.4-fold).

262

The effect of the deletion of PGUS C-terminal region on the enzyme expression level was

263

further investigated. PGUS and mutants D590-604, D591-604 were mostly expressed in soluble

264

form with few inclusion bodies upon comparing the protein contents of the supernatant and whole

265

cell (Fig. 5a). In addition, the expression levels of D590-604 and D591-604 were increased by 1.8-

266

fold compared to that of PGUS, which may be due to that the C-terminal region deletion prompts

267

the correct folding of PGUS, making it more easily expressed. We also investigated the growth

268

curve of three mutants (Fig. S3) and the growth rates of mutants D590-604 and D591-604 were

269

slower than that of wild-type cells, because the heavier expression burden of enzyme repressed the

270

cell growth. Notably, the C-terminal region deletion of PGUS was also beneficial for the enzyme

271

activity. The specific activities of purified mutants D590-604 and D591-604 were 1.4-fold and

272

2.7-fold higher than that of PGUS, respectively, which led the total activity to be 2.5-fold and 4.8-

273

fold higher than that of PGUS (Fig. 5b). Finally, we verified by native PAGE that the C-terminal

274

region deletion did not affect the oligomeric form of PGUS (Fig. S4).

275

The far-UV CD spectra of four mutants were similar to that of PGUS wide-type (Fig. S5).

276

The content distributions of α-helix and β-sheet did not show dramatic changes after the mutation,

277

as the β-sheet continued to be the main form with a content greater than 60% (Table S2). These

278

results indicated that the C-terminal region deletion had no significant effect on the secondary

279

structure. Fluorescence spectra were also analyzed to investigate the effect of deletion of C-

280

terminal region on the tertiary structure of PGUS (Fig. S6). PGUS and four mutants showed a 14


Page 14 of 39

Page 15 of 39


281

maximum absorbance at 331 nm, indicating that the mutation had no obvious effect on the tertiary

282

structure of the protein.

283

Investigating the regulating function of the C-terminal region of other GH2 GUSs

284

To investigate whether the C-terminal region has a regulating function for other GH2 enzymes,

285

we also deleted the C-terminal region of a bacterial GUS from E. coli (EGUS), generating two

286

mutants with different C-terminal lengths: D596-603 and D588-603 (the mutants were named by

287

the convention DX-X, where X-X demarcates the deleted residues). As shown in Fig. 6, EGUS

288

showed a tremendously higher thermostability than PGUS, with only a 3% activity loss after 100

289

min incubation at 70 °C. The C-terminal region deletion at various lengths had no clear effect on

290

the EGUS thermostability, since the mutants retained similar activity after 100 min heat-induced

291

denaturation, indicating that the C-terminal region performed no critical role in maintaining

292

stability. Nonetheless, the deletion of the partial C-terminal region (D597-603) increased the

293

soluble expression level by 1.6-fold without any loss of specific activity. Astonishingly, the

294

deletion of the whole C-terminal region of EGUS (D588-603) resulted in a 39.8% decrease in the

295

total expression level and a 49% decrease in the specific activity compared with PGUS, indicating

296

that the partial C-terminal region (residues 588-596) is necessary for maintaining the expression

297

level and activity of EGUS. To further verify its function, we introduced motif 588-596 of EGUS

298

to replace the whole C-terminal region of PGUS and generate a mutant named PGUSE (Fig. S7).

299

As expected, the soluble expression of PGUSE was double that of PGUS, indicating that motif

300

588-596 of EGUS prompts the correct folding not only of EGUS but also of PGUS. Nonetheless,

301

the thermostability of PGUSE was only slightly improved compared with that of PGUS. Therefore, 15



302

it can be concluded that the partial C-terminal region of EGUS (motif 588-596) was a functional

303

region regulating the protein expression. We also tried to heterologously express β-glucuronidase

304

from humans (HGUS) in E. coli, but the protein was mainly expressed in the form of inclusion

305

bodies. Nonetheless, a previous study has shown that HGUS is a lysosomal enzyme, and the whole

306

C-terminal region acts as a retention signal for phosphorylation and targeting the lysosome. When

307

the C-terminal region was deleted, 50% of the activity was lost, and 32-34% less phosphorylated

308

and targeting lysosome was observed.8, 50

309

Application of PGUS mutants in the biotransformation of glycyrrhizin

310

GL is a natural substrate for PGUS. GA, an important pharmaceutical ingredient, was produced

311

by hydrolyzing two glucuronic acid groups from the natural substrate GL via a two-step reaction

312

by PGUS (Fig. 7), and showed better activity and bioavailability. We established a fed-batch

313

process for preparing GA by GL hydrolysis mediated by PGUS and mutants D590-604 and D591-

314

604. As shown in Fig. 8, PGUS hydrolyzed GL in the initial 20 min, with a GL conversion of 66%,

315

and then, the reaction was paused which might be due to the substrate or product inhibition. This

316

point needs further investigation. There was no more GA formed at the final concentration of 0.39

317

g/L, even if more GL was added to the reaction system at 100 min. The GA yield was only 57.1%,

318

indicating that 42.9% byproduct GAMG was also formed due to that the PGUS activity was too

319

low to hydrolyze GAMG into GA. Under the same reaction conditions, for mutants D590-604 and

320

D591-604, GL was rapidly converted in 20 min, with the conversion reaching 88.1% and 84.7%,

321

respectively. The GA yield was 91.9% and 97.4%, respectively, indicating that 8.1% and 2.6%

322

GAMG was formed correspondingly. Then, 0.15 g fresh GL was fed into the reactants at 20 and 16


Page 16 of 39

Page 17 of 39


323

140 min, and the GA concentration continued to increase to 1.71 g/L and 1.58 g/L, respectively,

324

with a GL conversion of 83.6% and 78.1% at 200 min. In addition, the corresponding GA yields

325

were 81.2% and 81.6%, respectively, indicating that GA was the main product. The GL conversion

326

did not continue to increase after the GL feeding at 200 min, which may be due to the enzyme

327

activity was inhibited by the substrate or product. Therefore, two feeding times are optimal for the

328

reaction. All the above results indicate that PGUS mutants D590-604 and D591-604 are more

329

robust catalysts for the industrial application of GL hydrolysis.

330

DISCUSSION

331

Loop engineering such as deletion or swap has been increasingly adopted to manipulate the

332

stability and activity of enzymes. However, the precise targeting of hot-loops for engineering is

333

still quite challenging. In this study, computation-aided design on the basis of structural analysis

334

was employed to rationally identify a regulating C-terminal region that is critical for the low

335

stability of a GH2 glucuronidase PGUS. Then, several mutants with various deleted C-terminal

336

regions were designed that showed not only significantly improved kinetic and thermodynamic

337

stability but also enhanced expression level and activity. All these characteristics are critical for

338

the industrial application of enzymes. This indicates that our proposed method is quite effective in

339

targeting a flexible region for mutation. The deletion of the whole C-terminal region (D590-604,

340

D591-604) conferred better properties on PGUS than partial deletion (D595-604, D597-604),

341

indicating that the C-terminal region is redundant for PGUS to maintain its normal function.

342

Therefore, the C-terminal region deletion may yield a more compact overall structure, resulting in

343

a higher stability (Fig. 3). The thermostability of PGUS mutant D590-604 is higher than that of a 17



344

previously reported thermostable GUS from E. coli obtained by directed evolution, which had a

345

residual activity of approximately 90% after 30 min incubation at 70 °C, while the residual activity

346

of D590-604 was almost 100% under the same conditions.51, 52 In addition, all the PGUS mutants

347

showed similar pH profile as the wild-type (Fig. S8). The C-terminal region deletion was also more

348

favorable for protein folding, resulting in an increased protein expression level. Although the C-

349

terminal region was sterically far from the active site pocket, its deletion yielded an optimal active

350

site pocket conformation that was more favorable for substrate binding (lowering Km) and catalysis

351

(increasing kcat), thus resulting in the increase of the catalytic efficiency. This remote effect of

352

residues to tune catalytic behavior was achieved by conformational changes transmitted

353

throughout the protein backbone, which have also been reported in other enzymes.53-55

354

In addition, it was found that the N-terminal region of PGUS had no obvious impact on the

355

enzyme stability (Fig. 2), which is quite different from the case for enzymes from other GH

356

families such as GH5, GH10 and GH11, where the N-terminal region has been shown to play

357

critical roles in determining the enzyme stability and where a dramatic improvement in the enzyme

358

stability was achieved by deletion25 or exchange with a thermophilic counterpart.49 In addition, the

359

N-terminal coil of the GH10 xylanases was near the C-terminal region to initiate interaction, and

360

engineering this interaction can improve the enzyme thermostability.28, 56 All these results show

361

that the regulating function of the C-terminal region may be a unique feature of GH2 GUSs, which

362

is further evidenced by the fact that the C-terminal region was not conserved from the sequence

363

alignment of typical GUSs in GH2 (Fig. S9). This feature of the C-terminal feature is more

364

dramatic than that of the N-terminal in other reported GH families, since it regulates not only the 18


Page 18 of 39

Page 19 of 39


365

stability, but also the expression level and activity. To support this hypothesis, we also investigated

366

the regulating function of the C-terminal region of a GH2 bacterial GUS from E. coli (EGUS). The

367

deletion of its C-terminal region at various lengths did not result in dramatic improvement in the

368

enzyme stability and activity, which may be because that the stability of EGUS has already well

369

evolved to adapt to high temperature. However, the partial C-terminal region deletion (residues

370

597-603) caused an increased enzyme expression level, and the deletion of the whole C-terminal

371

region (residues 588-603) resulted in a decreased expression level and specific activity, indicating

372

that the partial C-terminal region (residues 588-596) was functional for normal enzyme expression

373

and secretion. Interestingly, we also found this region conferred an increased expression level to

374

PGUS (Fig. S7). HGUS was unsuccessfully expressed in E. coli due to the lack of glycosylation,

375

which is necessary for the proper folding and subunit assembly of HGUS.57 Nevertheless, the C-

376

terminal of HGUS has been demonstrated to have important physiological effects in post-

377

translation, lysosome targeting and maintaining enzyme activity. Therefore, the regulating

378

function of the C-terminal region is dramatically different for GUSs from bacteria, fungi and

379

humans.

380

All these results lead us to conclude that the C-terminal region may be an evolutionary feature

381

of GH2 GUSs. The C-terminal region (14 residues) of PGUS is evolved to be redundant, which

382

suppresses the catalytic capacity, and deleting this redundant part accelerates the evolution process

383

to prompt the folding of GUS into a more robust structure that displays a high expression level,

384

stability and activity. For EGUS, the C-terminal region (15 residues) is partially evolved to be

385

functional, so truncating this region (residues 597-603) improves the expression level with no 19



386

obvious changes in activity and stability, although this impact is not so dramatic for fungal GUS.

387

The functional part of the C-terminal region (residues 588-596) is necessary for the expression and

388

activity of EGUS. Notably, this part can also confer a significantly improved expression level to

389

PGUS (Fig. S7). The C-terminal region of human HGUS is evolved to be fully functional and

390

expanded to contain 20 residues, which are more than those of PGUS and EGUS. A previous study

391

has shown that deleting this part had various negative effects on the enzyme properties such as

392

decreasing the activity and post-translation degree and weakening the lysosome targeting.50 It is

393

anticipated that further examples of this tendency will become apparent in the near future, as more

394

GH2 GUSs from different species are characterized.

395

ASSOCIATED CONTENT

396

Supporting Information

397

The plasmid construction of GUS mutants; The SDS-PAGE, cell growth, native PAGE of PGUS

398

and mutants; The CD and fluorescence spectra of PGUS and mutants; Replacing the C-terminal

399

region of PGUS with partial C-terminal region of EGUS improved its expression level; The C-

400

terminal region of sequence alignment of typical GUSs in the GH2 family; The pH profile of

401

PGUS and mutants; The list of primers; The content of secondary structure.

402

ACKNOWLEDGMENT

403

This research was funded by grants from the National Natural Science Foundation of China (No.

404

21506011, No. 21425624, No. 21878021).

20


Page 20 of 39

Page 21 of 39

406 407 408


REFERENCES (1) Li-Weber, M. New therapeutic aspects of flavones: The anticancer properties of scutellaria and its main active constituents wogonin, baicalein and baicalin. Cancer Treat. Rev. 2009, 35, 57-68.

409

(2) Sverrisdóttir, E.; Lund, T. M.; Olesen, A. E.; Drewes, A. M.; Christrup, L. L.; Kreilgaard, M. A review of

410

morphine and morphine-6-glucuronide’s pharmacokinetic–pharmacodynamic relationships in experimental and

411

clinical pain. Eur. J. Pharm. Sci. 2015, 74, 45-62.

412

(3) Wu, Q.; Kroon, P. A.; Shao, H.; Needs, P. W.; Yang, X. Differential effects of quercetin and two of its derivatives,

413

isorhamnetin and isorhamnetin-3-glucuronide, in inhibiting the proliferation of human breast-cancer MCF-7 cells. J.

414

Agric. Food Chem. 2018, 66, 7181-7189.

415 416 417 418

(4) Lv, B.; Sun, H.; Huang, S.; Feng, X.; Jiang, T.; Li, C. Structure-guided engineering of the substrate specificity of a fungal β-glucuronidase toward triterpenoid saponins. J. Biol. Chem. 2018, 293, 433-443. (5) Song, X.; Jiang, Z.; Li, L.; Wu, H. Immobilization of β-glucuronidase in lysozyme-induced biosilica particles to improve its stability. Front. Chem. Sci. Eng. 2014, 8, 353-361.

419

(6) Sakurama, H.; Kishino, S.; Uchibori, Y.; Yonejima, Y.; Ashida, H.; Kita, K.; Takahashi, S.; Ogawa, J. beta-

420

Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30. Appl.

421

Microbiol. Biotechnol. 2014, 98, 4021-4032.

422

(7) Pollet, R. M.; D'Agostino, E. H.; Walton, W. G.; Xu, Y.; Little, M. S.; Biernat, K. A.; Pellock, S. J.; Patterson,

423

L. M.; Creekmore, B. C.; Isenberg, H. N.; Bahethi, R. R.; Bhatt, A. P.; Liu, J.; Gharaibeh, R. Z.; Redinbo, M. R. An

424

atlas of beta-glucuronidases in the human intestinal microbiome. Structure 2017, 25, 1-11.

425 426

(8) Jain, S.; Drendel, W. B.; Chen, Z. W.; Mathews, F. S.; Sly, W. S.; Grubb, J. H. Structure of human betaglucuronidase reveals candidate lysosomal targeting and active-site motifs. Nat. Struct. Biol. 1996, 3, 375-381. 21



427

(9) Wallace, B. D.; Wang, H.; Lane, K. T.; Scott, J. E.; Orans, J.; Koo, J. S.; Venkatesh, M.; Jobin, C.; Yeh, L. A.;

428

Mani, S.; Redinbo, M. R. Alleviating cancer drug toxicity by inhibiting a bacterial enzyme. Science 2010, 330, 831-

429

5.

430

(10) Wallace, B. D.; Roberts, A. B.; Pollet, R. M.; Ingle, J. D.; Biernat, K. A.; Pellock, S. J.; Venkatesh, M. K.;

431

Guthrie, L.; O'Neal, S. K.; Robinson, S. J.; Dollinger, M.; Figueroa, E.; McShane, S. R.; Cohen, R. D.; Jin, J.; Frye,

432

S. V.; Zamboni, W. C.; Pepe-Ranney, C.; Mani, S.; Kelly, L.; Redinbo, M. R. Structure and inhibition of microbiome

433

beta-glucuronidases essential to the alleviation of cancer drug toxicity. Chem Biol. 2015, 22, 1238-1249.

434

(11) Muderrisoglu, C.; Sargin, S.; Yesil-Celiktas, O. Application of beta-glucuronidase-immobilised silica gel

435

formulation to microfluidic platform for biotransformation of beta-glucuronides. Biotechnol. Lett. 2018, 40, 773-780.

436

(12) Xu, Y.; Feng, X.; Jia, J.; Chen, X.; Jiang, T.; Rasool, A.; Lv, B.; Qu, L.; Li, C. A novel beta-glucuronidase from

437

Talaromyces pinophilus Li-93 precisely hydrolyzes glycyrrhizin into glycyrrhetinic acid 3-O-mono-beta-D-

438

glucuronide. Appl. Environ. Microbiol. 2018, 84, e00755-18.

439 440 441 442 443 444 445 446 447

(13) Yu, H. R.; Huang, H. Engineering proteins for thermostability through rigidifying flexible sites. Biotechnol. Adv. 2014, 32, 308-315. (14) Liu, Y.; Chen, B.; Wang, Z.; Liu, L.; Tan, T. Functional characterization of a thermostable methionine adenosyltransferase from Thermus thermophilus HB27. Front. Chem. Sci. Eng. 2016, 10, 238-244. (15) Denard, C. A.; Ren, H. Q.; Zhao, H. M. Improving and repurposing biocatalysts via directed evolution. Curr. Opin. Chem. Biol. 2015, 25, 55-64. (16) Yamada, R.; Higo, T.; Yoshikawa, C.; China, H.; Ogino, H. Improvement of the stability and activity of the BPO-A1 haloperoxidase from Streptomyces aureofaciens by directed evolution. J. Biotechnol. 2014, 192, 248-254. (17) Wu, I.; Arnold, F. H. Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose 22


Page 22 of 39

Page 23 of 39

448 449 450


efficiently at elevated temperatures. Biotechnol. Bioeng. 2013, 110, 1874-1883. (18) Cao, L. C.; Chen, R.; Xie, W.; Liu, Y. H. Enhancing the thermostability of feruloyl esterase EstF27 by directed evolution and the underlying structural basis. J. Agric. Food Chem. 2015, 63, 8225-8233.

451

(19) Trollope, K. M.; Gorgens, J. F.; Volschenk, H. Semirational directed evolution of loop regions in Aspergillus

452

japonicus beta-fructofuranosidase for improved fructooligosaccharide production. Appl. Environ. Microbiol. 2015, 81,

453

7319-7329.

454

(20) Xu, B. L.; Dai, M. H.; Chen, Y. H.; Meng, D. H.; Wang, Y. S.; Fang, N.; Tang, X. F.; Tang, B. Improving the

455

thermostability and activity of a thermophilic subtilase by incorporating structural elements of its psychrophilic

456

counterpart. Appl. Environ. Microbiol. 2015, 81, 6302-6313.

457

(21) Niu, C. F.; Yang, P. L.; Luo, H. Y.; Huang, H. Q.; Wang, Y. R.; Yao, B. Engineering of Yersinia phytases to

458

improve pepsin and trypsin resistance and thermostability and application potential in the food and feed industry. J.

459

Agric. Food Chem. 2017, 65, 7337-7344.

460 461

(22) Huang, J.; Jones, B. J.; Kazlauskas, R. J. Stabilization of an α/β-hydrolase by introducing proline residues: Salicylic acid binding protein 2 from tobacco. Biochemistry 2015, 54, 4330-4341.

462

(23) Nestl, B. M.; Hauer, B. Engineering of flexible loops in enzymes. ACS Catal. 2014, 4, 3201-3211.

463

(24) Yang, H. Q.; Liu, L.; Shin, H. D.; Chen, R. R.; Li, J. H.; Du, G. C.; Chen, J. Integrating terminal truncation and

464

oligopeptide fusion for a novel protein engineering strategy to improve specific activity and catalytic efficiency:

465

alkaline alpha-amylase as a case study. Appl. Environ. Microbiol. 2013, 79, 6429-6438.

466

(25) Damnjanovic, J.; Nakano, H.; Iwasaki, Y. Deletion of a dynamic surface loop improves stability and changes

467

kinetic behavior of phosphatidylinositol-synthesizing Streptomyces phospholipase D. Biotechnol. Bioeng. 2014, 111,

468

674-682. 23



469 470 471 472 473 474

(26) Reich, S.; Kress, N.; Nest, B. M.; Hauer, B. Variations in the stability of NCR ene reductase by rational enzyme loop modulation. J. Struct. Biol. 2014, 185, 228-233. (27) Boone, C. D.; Rasi, V.; Tu, C.; McKenna, R. Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II. FEBS J. 2015, 282, 1445-1457. (28) Song, L.; Tsang, A.; Sylvestre, M. Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids. Biotechnol. Bioeng. 2015, 112, 1081-1091.

475

(29) Dong, Y. H.; Li, J. F.; Hu, D.; Yin, X.; Wang, C. J.; Tang, S. H.; Wu, M. C. Replacing a piece of loop-structure

476

in the substrate-binding groove of Aspergillus usamii beta-mannanase, AuMan5A, to improve its enzymatic properties

477

by rational design. Appl. Microbiol. Biotechnol. 2016, 100, 3989-3998.

478 479 480 481 482 483

(30) Silva, P. J.; Ramos, M. J. Computational characterization of the substrate-binding mode in coproporphyrinogen III oxidase. J. Phys. Chem. B 2011, 115, 1903-1910. (31) Chen, Q.; Luan, Z. J.; Cheng, X.; Xu, J. H. Molecular dynamics investigation of the substrate binding mechanism in carboxylesterase. Biochemistry 2015, 54, 1841-8. (32) Niu, C.; Zhu, L.; Zhu, P.; Li, Q. Lysine-based site-directed mutagenesis increased rigid β-sheet structure and thermostability of mesophilic 1,3–1,4-β-glucanase. J. Agric. Food Chem. 2015, 63, 5249-5256.

484

(33) Huang, J.; Xie, D.-F.; Feng, Y. Engineering thermostable (R)-selective amine transaminase from Aspergillus

485

terreus through in silico design employing B-factor and folding free energy calculations. Biochem. Biophys. Res.

486

Commun. 2017, 483, 397-402.

487

(34) Yu, J. Y.; Ha, J. Y.; Kim, K. M.; Jung, Y. S.; Jung, J. C.; Oh, S. Anti-inflammatory activities of licorice extract

488

and its active compounds, glycyrrhizic acid, liquiritin and liquiritigenin, in BV2 cells and mice liver. Molecules 2015,

489

20, 13041-13054. 24


Page 24 of 39

Page 25 of 39

490 491 492 493 494 495 496 497 498 499


(35) Han, S. W.; Sun, L.; He, F.; Che, H. L. Anti-allergic activity of glycyrrhizic acid on IgE-mediated allergic reaction by regulation of allergy-related immune cells. Sci. Rep. 2017, 7. (36) Su, X. T.; Wu, L.; Hu, M. M.; Dong, W. X.; Xu, M.; Zhang, P. Glycyrrhizic acid: A promising carrier material for anticancer therapy. Biomed. Pharmacother. 2017, 95, 670-678. (37) Zhao, Y.; Lv, B.; Feng, X.; Li, C. Perspective on biotransformation and de novo biosynthesis of licorice constituents. J. Agric. Food Chem. 2017, 65, 11147-11156. (38) Akao, T. Differences in the metabolism of glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid monoglucuronide by human intestinal flora. Biol. Pharm. Bull. 2000, 23, 1418-1423. (39) Reetz, M. T.; Carballeira, J. D. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2007, 2, 891-903.

500

(40) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular dynamics. J. Mol. Graph. 1996, 14, 33-38.

501

(41) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of simple potential

502 503 504 505 506

functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926-935. (42) Darden, T.; York, D.; Pedersen, L. Particle mesh ewald - An n.log(n) method for ewald sums in large systems. J. Chem. Phys. 1993, 98, 10089-10092. (43) Bradford, M. M. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254.

507

(44) Pace, C. N. Measuring and increasing protein stability. Trends Biotechnol. 1990, 8, 93-98.

508

(45) Tan, N. Y.; Bailey, U. M.; Jamaluddin, M. F.; Mahmud, S. H. B.; Raman, S. C.; Schulz, B. L. Sequence-based

509 510

protein stabilization in the absence of glycosylation. Nat. Commun. 2014, 5. (46) Reetz, M. T.; Carballeira, J. D.; Vogel, A. Iterative saturation mutagenesis on the basis of B factors as a strategy 25



511

for increasing protein thermostability. Angewandte Chemie 2006, 118, 7909-7915.

512

(47) Yang, T. C.; Legault, S.; Kayiranga, E. A.; Kumaran, J.; Ishikawa, K.; Sung, W. L. The N-terminal beta-sheet

513

of the hyperthermophilic endoglucanase from Pyrococcus horikoshii is critical for thermostability. Appl. Environ.

514

Microbiol. 2012, 78, 3059-3067.

515

(48) Dumon, C.; Varvak, A.; Wall, M. A.; Flint, J. E.; Lewis, R. J.; Lakey, J. H.; Morland, C.; Luginbuhl, P.; Healey,

516

S.; Todaro, T.; DeSantis, G.; Sun, M.; Parra-Gessert, L.; Tan, X. Q.; Weiner, D. P.; Gilbert, H. J. Engineering

517

hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure. J. Biol. Chem. 2008,

518

283, 22557-22564.

519

(49) Gao, S. J.; Wang, J. Q.; Wu, M. C.; Zhang, H. M.; Yin, X.; Li, J. F. Engineering hyperthermostability into a

520

mesophilic family 11 xylanase from Aspergillus oryzae by in silico design of N-terminus substitution. Biotechnol.

521

Bioeng. 2013, 110, 1028-1038.

522

(50) Islam, M. R.; Grubb, J. H.; Sly, W. S. C-terminal processing of human beta-glucuronidase: The propeptide is

523

required for full expression of catalytic activity, intracelluar retention, and proper phosphorylation. J. Biol. Chem.

524

1993, 268, 22627-22633.

525

(51) Xiong, A.-S.; Peng, R.-H.; Liu, J.-G.; Zhuang, J.; Qiao, Y.-S.; Xu, F.; Cai, B.; Zhang, Z.; Chen, J.-M.; Yao,

526

Q.-H. High efficiency and throughput system in directed evolution in vitro of reporter gene. Appl. Microbiol.

527

Biotechnol. 2007, 74, 160-168.

528

(52) Xiong, A.-S.; Peng, R.-H.; Cheng, Z.-M.; Li, Y.; Liu, J.-G.; Zhuang, J.; Gao, F.; Xu, F.; Qiao, Y.-S.; Zhang, Z.;

529

Chen, J.-M.; Yao, Q.-H. Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability.

530

Protein Eng. Des. Sel. 2007, 20, 319-325.

531

(53) Tiwari, M. K.; Singh, R. K.; Singh, R.; Jeya, M.; Zhao, H. M.; Lee, J. K. Role of conserved glycine in zinc26


Page 26 of 39

Page 27 of 39

532 533 534


dependent medium chain dehydrogenase/reductase superfamily. J. Biol. Chem. 2012, 287, 19429-19439. (54) Tiwari, M. K.; Kalia, V. C.; Kang, Y. C.; Lee, J. K. Role of a remote leucine residue in the catalytic function of polyol dehydrogenase. Mol. BioSyst. 2014, 10, 3255-3263.

535

(55) Jimenez-Oses, G.; Osuna, S.; Gao, X.; Sawaya, M. R.; Gilson, L.; Collier, S. J.; Huisman, G. W.; Yeates, T. O.;

536

Tang, Y.; Houk, K. N. The role of distant mutations and allosteric regulation on LovD active site dynamics. Nat.

537

Chem. Biol. 2014, 10, 431-436.

538

(56) Mahanta, P.; Bhardwaj, A.; Kumar, K.; Reddy, V. S.; Ramakumar, S. Structural insights into N-terminal to C-

539

terminal interactions and implications for thermostability of a (beta/alpha)(8)-triosephosphate isomerase barrel

540

enzyme. FEBS J. 2015, 282, 3543-3555.

541 542

(57) Shipley, J. M.; Grubb, J. H.; Sly, W. S. The role of glycosylation and phosphorylation in the expression of active human beta-glucuronidase. J. Biol. Chem. 1993, 268, 12193-12198.

543 544

27



545

List of Figure Legends

546

Figure 1 (a) B-factor and (b) root mean squared fluctuations (RMSF) measured during a 95-ns

547

MD simulation. (c) Ribbon diagram of the conformation of the C-terminal region reconstructed by

548

computational modeling. The C-terminal region is displayed in green, and the adjacent α-helix is

549

shown in orange.

550

Figure 2 Root mean squared deviation (RMSD) of mutants with deleted C-terminal region (a) and

551

N-terminal region (b) measured during a 95-ns MD simulation.

552

Figure 3 Thermostability of PGUS and mutants at (a) 65 °C and (b) 70 °C. Values are averages of

553

three independent replicates; error bars represent average ± one standard deviation.

554

Figure 4 Thermodynamic stability of PGUS and mutants: (a) D590-604, (b) D591-604, (c) D595-

555

604, and (d) D597-604, as determined by monitoring the intrinsic fluorescence of the proteins

556

during their incubation with guanidinium and then fitting these data to the two-state model of

557

protein folding.

558

Figure 5 (a) Expression level of PGUS and mutants D590-604 and D591-604 as analyzed by SDS-

559

PAGE. M: marker; lane 1: PGUS-supernatant; lane 2: PGUS-whole cell; lane 3: D590-604-

560

supernatant; lane 4: D590-604-whole cell; lane 5:D591-604-supernatant; lane 6: D591-604-whole

561

cell; lane 7: No-induction whole cell. The loading volume of the supernatant and whole cell volume

562

was 4 μL. (b) Specific activity of PGUS and mutants characterized by pNPG hydrolysis. Values

563

are averages of three independent replicates; error bars represent average ± one standard deviation. 28


Page 28 of 39

Page 29 of 39


564

Figure 6 Effect of the C-terminal region deletion on the thermostability at 70 °C (left panel) and

565

expression level, specific activity (right panel) of EGUS. The thermostability and specific activity

566

tests were conducted in triplicate, and the errors represent mean ± one standard deviation. For

567

SDS-PAGE, M: marker; lane 1: EGUS-supernatant; lane 2: EGUS-whole cell; lane 3: D588-603-

568

supernatant; lane 4: D588-603-whole cell; lane 5: D597-603-supernatant; lane 6: D597-603-whole

569

cell. The loading volume of the supernatant and whole cell was 4 μL.

570

Figure 7 Scheme of two-step hydrolysis of glycyrrhizin (GL) into glycyrrhetic acid (GA)

571

mediated by PGUS.

572

Figure 8 Fed-batch process for preparing glycyrrhetinic acid (GA) by glycyrrhizin (GL)

573

hydrolysis mediated by PGUS and mutants D590-604 and D591-604. (a) GA concentration, (b)

574

GL conversion and (c) GA yield as a function of reaction time. For reactions mediated by PGUS,

575

GL was fed at 100 min. For reaction mediated by D590-604 and D591-604, GL was fed at 20 min,

576

140 min and 200 min. Values are averages of three independent replicates; error bars represent

577

average ± one standard deviation.

578

29



579

580

Page 30 of 39

Table 1 Kinetic parameters of PGUS and mutants Enzyme

Km (mM)

kcat (s-1)

kcat/Km (s-1 mM-1)

PGUS D590-604 D591-604 D595-604 D597-604

1.2 ± 0.09 0.5 ± 0.03 1.3 ± 0.1 1.4 ± 0.07 0.6 ± 0.02

162.4 ± 0.6 160.6 ± 8.5 427.1 ± 6.0 313.1 ± 2.2 193.6 ± 1.9

135.3 321.2 328.5 223.6 322.7

Results are from triplicate measurement, and uncertainties are denoted as the average ± one standard deviation.

581 582 583 584 585 586 587 588

30


Page 31 of 39


Figure 1 192x494mm (600 x 600 DPI)



Figure 2 133x223mm (600 x 600 DPI)


Page 32 of 39

Page 33 of 39


Figure 3 136x232mm (600 x 600 DPI)



Figure 4 121x103mm (600 x 600 DPI)


Page 34 of 39

Page 35 of 39


Figure 5 66x31mm (600 x 600 DPI)





Page 36 of 39

Page 37 of 39





Figure 8 199x499mm (600 x 600 DPI)


Page 38 of 39

Page 39 of 39


Graphic for table of contents 47x26mm (600 x 600 DPI)


Computation-Aided Rational Deletion of C-Terminal Region Improved

Recommend Documents