Insight into the Thermophilic Mechanism of a Glycoside Hydrolase

Subscriber access provided by University of Winnipeg Library

Functional Structure/Activity Relationships

Insight into the Thermophilic Mechanism of a Glycoside Hydrolase Family 5 #-Mannanase Weina Liu, Tao Tu, Yuan Gu, Yuan Wang, Fei Zheng, Jie Zheng, Yaru Wang, Xiaoyun Su, Bin Yao, and Huiying luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04860 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 7, 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 45

Journal of Agricultural and Food Chemistry

1

Insight into the Thermophilic Mechanism of a Glycoside Hydrolase Family 5

2

β-Mannanase

3 4

Weina Liu#, Tao Tu#, Yuan Gu, Yuan Wang, Fei Zheng, Jie Zheng, Yaru Wang,

5

Xiaoyun Su, Bin Yao*, Huiying Luo*

6 7

Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed

8

Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100086,

9

People’s Republic of China

10 11 12

#

13

*

14

Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural

15

Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China. Tel.: +86

16

10 82106053; fax: +86 10 82106054. E-mail addresses: [email protected] (H.

17

Luo), [email protected] (B. Yao).

WL and TT contributed equally to this work.

Corresponding authors. Address: Key Laboratory for Feed Biotechnology of the

18 19 20 21 22 1

ACS Paragon Plus Environment


23

ABSTRACT: To study the molecular basis for thermophilic -mannanase of

24

glycoside hydrolase family 5, two -mannanases, TlMan5A and PMan5A, from

25

Talaromyces leycettanus JCM12802 and Penicillium sp. WN1 were used as models.

26

The four residues, His112 and Phe113 located near the antiparallel -sheet at the

27

barrel bottom and Leu375 and Ala408 from loop 7 and loop 8 of PMan5A, were

28

inferred to be key thermostability contributors through module substitution, truncation,

29

and site-directed mutagenesis. The effects of these four residues on the thermal

30

properties followed the order H112Y>A408P>L375H>F113Y and were strongly

31

synergetic. These results were interpreted structurally using molecular dynamics (MD)

32

simulations, which showed that improved hydrophobic interactions in the inner wall

33

of the -barrel and the rigidity of loop 8 were caused by the outside domain of the

34

barrel bottom and proline, respectively. The TIM barrel bottom and four specific

35

residues responsible for the thermostability of GH5 -mannanases were elucidated.

36

KEYWORDS: Glycoside hydrolase family 5 (GH5); -mannanase; thermostability;

37

site-directed mutagenesis; molecular dynamics (MD) simulation

2


Page 2 of 45

Page 3 of 45


38 39

INTRODUCTION Thermophilic enzymes with inherent stability and higher activity at high

40

temperatures compared with mesophilic and psychrophilic enzymes are of great

41

significance in industrial and biotechnological applications.1 Other advantages,

42

including a higher resistance to harsh solution conditions (such as pH, cosolvent, etc.),

43

higher reaction rates and conversion efficiencies at high temperatures, lower

44

microbial contamination,1,2 and lower energy requirements3 have been presented by

45

thermophilic enzymes. The amino acid interactions of proteins, such as hydrophobic

46

interactions, salt bridges, disulfide bridges, π–π interactions, cation–π interactions,

47

and interaction networks, have been reported to account for their structural stability

48

and thermostability.4 For example, the Candida antarctica lipase B, CalB, showed

49

enhanced kinetic stability when an extra hydrogen bond network was formed.5

50

Furthermore, a modified GH5 -mannanase with additional salt bridges and ion

51

triplets resulted in a higher optimal temperature.6 A large number of experimental and

52

computational studies have also demonstrated that global structures and dynamics

53

play significant roles in stabilizing protein structures.4 Loop structures with free

54

conformation are shorter in thermophilic proteins than in their mesophilic

55

homologues, and are related to protein flexibility.7,8 Thermophilic enzymes usually

56

contain more proline than their mesophilic homologues, owing to its higher native

57

state conformational entropy, as well as arginine and tyrosine.9 Together, these factors

58

make crucial contributions to enzyme thermal stability.

59

Endo--mannanase (EC 3.2.1.78), which catalyzes the random hydrolysis of 3



60

-1,4-glucosidic linkages in various polymannose backbones, is a well-known

61

biotechnological hemicellulase.10 Owing to amino acid sequence similarities, most

62

endo--mannanases are confined to glycoside hydrolase (GH) families 5, 26, and 113

63

(http//:www.cazy.org).11 These enzymes belong to clan GH-A, which share similar

64

(/)8 barrel-fold spatial architectures and follow a double displacement catalysis

65

mechanism7,12 in which two strictly conserved glutamate residues serve as a general

66

acid/base and nucleophile.13 This canonical (/)8 barrel-fold is characterized by a

67

central -barrel formed by eight major parallel -strands and surrounded by eight

68

major -helices, with the -strands and -helices connected by - or -loops.14,15

69

Structural analysis has shown that (/)8-barrel enzymes have a “catalytic face” at the

70

C-terminal ends of the -strands and a “stability face” of residues located on the

71

-loops at the bottom of the cylindric -barrel.14

72

GH5 -mannanases often display multiple modular architectures, including a

73

carbohydrate-binding module (CBM), a catalytic domain (CD), and some additional

74

functional modules.16 The CBM not only contributes to substrate bonding, but also

75

optimizes the molecular structure for high temperatures.17–19 However, its

76

contribution to protein thermostability is irregular. Deletion of the CBM1 module

77

from GH5 -mannanase from T. leycettanus JCM12802 resulted in worse thermal

78

tolerance at 80 °C.20 The thermostability of GH5 -mannanase (AuMan5A) from A.

79

usamii YL-01-78 has been improved by fusion with CBM1 of the cellobiohydrolase I

80

derived from T. reesei (TrCBH I).21 Recent observations have also provided

81

unconventional evidence that CBM1-truncated A. nidulans XZ3 -mannanase 4


Page 4 of 45

Page 5 of 45


82

(Man5XZ3) shows improved thermostability.22 Furthermore, the linker region,

83

usually between the CBM and CD, can serve as a stabilizing factor because the

84

truncated variant Man5ΔCL without CBM1 and linker shows weaker

85

thermostability.22 The N- and C-terminal regions have also been identified as key

86

factors in protein rigidity, whose removal significantly reducing the thermostability of

87

endoglucanase EGPh.23 Cyclic residues, such as proline and phenylalanine, can also

88

enhance the protein rigidity of several (/)8-barrel enzymes24, 25 or hydrophobic

89

interactions of cellulase Cel7A.26 These elements, individually or in combination,

90

play a role in protein thermostability.

91

Thermophilic mannanases have attracted global attention owing to their broad

92

applications in food processing, kraft pulp delignification, and feed production.27–29

93

With increasing demand for thermophilic -mannanases, protein engineering

94

strategies such as directed evolution and structure-guided rational design have been

95

employed to develop new endo--mannanases with high thermostability. Dong et al.30

96

employed a loop-structure substitution strategy using megaprimer PCR to improve the

97

Tm value and half-life of A. usamii -mannanase by 12.1 °C and 48-fold, respectively.

98

Many studies have been conducted to determine the thermostable mechanism of

99

endo--mannanases by analyzing their molecular, biochemical, and biological

100

properties.31 To identify the key factors related to the thermostability of GH5

101

endo--mannanases, a novel endo-mannanase of GH5 from Penicillium sp. WN1,

102

PMan5A, and its homologue from Talaromyces leycettanus JCM12802, TlMan5A,20

103

were evaluated in this mechanism study. These enzymes shared 73% sequence 5



104

identity, but had different thermal properties. Through systematic and rational design,

105

chimeras of PMan5A and TlMan5A and mutants with replacement key domains or

106

residues were constructed. The local structures were analyzed using molecular

107

dynamics (MD) simulations, and the underlying thermostability mechanism of GH5

108

-mannanases was elucidated.

109

MATERIALS AND METHODS

110

Strains, kits, and chemicals. Escherichia coli TransІ-T1 (TransGen, Beijing,

111

China) and Pichia pastoris GS115 (Invitrogen, Carlsbad, CA) were used as the hosts

112

for plasmid construction and heterologous expression, respectively. FastPfu Fly DNA

113

Polymerase and a Fast Mutagenesis System kit were purchased from TransGen

114

Biotech (Catalog number: AP231-01 and FM111-01). The restriction enzymes and T4

115

DNA ligase were purchased from ThermoFisher Scientific (Catalog number:

116

15224017, Waltham, MA). Standard D-(+)-mannose and substrate locust bean gum

117

(LBG) were purchased from Sigma-Aldrich (Catalog number: M2069 and 62631, St.

118

Louis, MO). Other chemicals were of analytical grade and commercially available.

119

Gene cloning. A novel GH5 endo--mannanase-encoding gene, PMan5A, was

120

identified in Penicillium sp. strain WN1 (CGMCC5131 of the China General

121

Microbiological Culture Collection Center, Beijing, China). The total RNA of the

122

fungus was isolated and purified after three days growth in mannanase-producing

123

medium20 at 45 °C using the SV Total RNA Isolation System (Catalog number:

124

Z3101, Promega, Madison, WI). cDNAs were synthesized in vitro using the ReverTra

125

Ace--TM kit (Catalog number: FSQ-101, Toyobo, Osaka, Japan). The gene 6


Page 6 of 45

Page 7 of 45


126

fragment coding for mature PMan5A was amplified with specific oligonucleotide

127

primer sets PMan5A_f and PMan5A_r (Table S1) and ligated (T4 DNA ligase) into

128

plasmid pPIC9. The resulting construct (pPIC9-PMan5A) was analyzed by colony

129

PCR and sequenced at the 5′ and 3′ ends to verify the correct insertion.

130

Rational design and construction of the recombinant plasmids. To gain

131

further insight into the thermophilic mechanism of GH5 -mannanases, the amino

132

acid sequences and secondary structures of PMan5A and TlMan5A were aligned

133

using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and ESPript 3.0

134

(http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) software. Each protein was divided

135

into four domains of intact structure. Recombinant plasmids pPIC9-TlMan5A 20 and

136

pPIC9-PMan5A were then used for mutant construction. Gene fragments coding for

137

the chimeric proteins were then obtained by overlap extension PCR with specific

138

primers (Table S1). After EcoRI and NotI digestion, the products were cloned into

139

vector pPIC9 (Invitrogen). Site-directed mutagenesis using plasmids pPIC9-PMan5A

140

or pPIC9-M1 as the template was performed by PCR according to manufacturer

141

instructions (TransGen). The PCR products were digested by DpnI (#R0176V, New

142

England Biolabs, Hitchin, UK) to remove the methylated plasmid template. All

143

constructs were then transformed into E. coli TransI-T1 competent cells by heat shock

144

and positive clones were validated by DNA sequencing.

145

Enzyme expression and purification. Recombinant plasmids harboring target

146

gene fragments were linearized by DraI and then electroporated into P. pastoris

147

GS115 competent cells. Enzyme induction was performed according to the Operating 7



148

Manual of the EasySelect Pichia Expression Kit (Invitrogen). Specifically, the His+

149

transformants were screened on minimal dextrose medium agar plates (MD; 2%

150

glucose and 2% agarose), with positive transformants confirmed by their enzymatic

151

activities at shake-tube level. Transformants showing the highest mannanase activity

152

were selected for growth in buffered glycerol complex medium (BMGY; 400 mL) for

153

cell propagation and buffered methanol complex medium (BMMY; 200 mL)

154

containing 0.5% (v/v) methanol at 30 °C and 200 rpm for 48 h. The culture

155

supernatants were harvested at 12,000 ×g for 10 min at 4 °C.

156

The cell-free cultures were purified by anion exchange chromatography on a

157

HiTrap Q Sepharose XL 5-mL fast protein liquid chromatography (FPLC) column

158

(GE Healthcare, Uppsala, Sweden) equilibrated with three column volumes of 20 mM

159

citric acid–Na2HPO4 (buffer A; pH 7.2). Proteins were eluted with a linear gradient of

160

sodium chloride (0–1.0 M). Fractions were collected and pooled based on their

161

mannanase activity and SDS-PAGE analysis. Endo--N-acetylglucosaminidase H

162

(Endo H; New England Biolabs) was used to remove N-glycan at 37 °C overnight.

163

The concentrations of the purified enzymes were determined by the Bradford method

164

using bovine serum albumin as the standard.

165

Enzyme activity assay. To determine the activity of -mannanase, the

166

3,5-dinitrosalicylic acid (DNS) method32 was used to detect reducing sugars released

167

from LBG with D-(+)-mannose as a standard. Reaction mixtures containing 900 L of

168

0.5% LBG (w/v) in McIlvaine buffer (100 mM citric acid and 200 mM Na2HPO4, pH

169

5.0) and opportunely diluted enzyme (100 L) were incubated for 10 min at the 8


Page 8 of 45

Page 9 of 45


170

optimal temperature. DNS reagent (1.5 mL) was added to terminate the reactions.

171

After boiling for 5 min in a water bath, the amount of released reducing sugars was

172

determined by measuring the absorbance at 540 nm. One unit of -mannanase activity

173

was defined as the amount of enzyme producing 1 mol of reducing sugar (as

174

mannose) in 1 min under the assay conditions (pH 4.5 or 5.0, 7085 °C, and 10 min).

175

Characterization of purified recombinant enzymes. The temperature optima

176

(Topt) were determined at temperatures of 40–90 °C in McIlvaine buffer (pH 5.0 or

177

4.5) for 10 min and the pH-adaptive profiles were analyzed in pH 2.2–8.0 buffers at

178

the optimal temperature for 10 min. The thermal stability was tested by preincubating

179

the diluted enzyme (approximately 100 g/mL) at 40–80 °C for 30 min and the pH

180

stability was assayed after preincubating the enzyme (approximately 100 g/mL) in

181

different buffers (pH 1.0–12.0) at 37 °C for 1 h. The residual activities were then

182

assessed under standard conditions. The buffers used were as follows: glycine–HCl

183

(100 mM, pH 1.03.0), McIlvaine buffer (100 mM, pH 2.28.0), Tris-HCl (100 mM,

184

pH 8.09.0), and glycine–NaOH (100 mM, pH 9.012.0).

185

T50 is the temperature at which an enzyme retains 50% of its initial activity after

186

incubation for 30 min. The half-life (t1/2) is the preincubation time in which an

187

enzyme loses 50% activity. To determine the thermal inactivation at T50 and t1/2, the

188

purified enzymes (100 g/mL, pH 5.0) were incubated without substrate at

189

temperatures of 60–85°C for 30 min, or incubated at 75 and 80 °C for 0–180 min. The

190

initial and residual activities were measured at pH 5.0 and the temperature optimum

191

for 10 min. All tests were performed in triplicate. 9



192

Determination of melting temperature (Tm). Differential scanning calorimetry

193

(DSC; TA Instruments, New Castle, DE) was used to determine the Tm values of

194

wild-type and mutant enzymes. Enzymes were dissolved in 10 mM McIlvaine buffer

195

(pH 7.2) at a concentration of 0.2 mg/mL. All samples were degassed for 10 min and

196

then scanned from 25 to 100 °C at a heating rate of 1 °C/min. The thermal

197

inactivation of each enzyme was irreversible. All tests were performed in triplicate.

198

Kinetics assays. The initial velocities of each enzyme toward various

199

concentrations of LBG (0.375–5 mg/mL) were measured under optimal conditions.

200

The Km, Vmax and kcat values were then determined using a nonlinear regression

201

algorithm in Graph-Pad Prism 5.0 software (La Jolla, CA). The catalytic efficiency

202

(kcat/Km) was also calculated.

203

MD simulations. The dynamic properties of PMan5A and its mutant

204

H112Y/F113Y/L375H/A408P were compared to elucidate the possible

205

thermostability mechanism. To keep each system at the same initial configuration, the

206

apo monomers were submitted to in silico mutagenesis using Discovery Studio 2017

207

software (Accelrys, San Diego, CA), the crystal structure of -mannanase AnManBK

208

(3WH9, 68% identity) was used as the template.33 MD simulations were performed

209

using the AMBER 14 software package along with the AMBER99SB force field.34

210

The initial structures were placed in a dodecahedral box filled with TIP3P water

211

molecules and 1-nm padding between the protein atoms and box sides. Sodium or

212

chloride ions were placed around the system to neutralize the charge. To remove

213

potentially poor contacts between the solute and solvent, the water molecules/ions 10


Page 10 of 45

Page 11 of 45


214

were resolved to minimize energy in 10,000 steps, followed by 10,000 steps for the

215

side chains of the protein and 10,000 steps for the whole system. After energy

216

minimization, the systems were gradually heated from 0 to 300 K over 100 ps,

217

followed by 50-ns production of MD simulations with a time step of 2 fs at a

218

temperature of 300 K and pressure of 1.0 atm controlled by the Langevin algorithm.

219

Long-range electrostatic interactions were analyzed using the particle-mesh Ewald

220

(PME) method.35 Nonbonded van der Waals interactions were calculated with a cutoff

221

of 1.0 nm. Bonds involving hydrogen atoms were constrained by the SHAKE

222

algorithm.36

223

RESULTS

224

Enzyme production and characterization. The PMan5A-encoding gene

225

(GenBank accession no. MH675997) contains 1308 base pairs, which codes for a

226

polypeptide of 435 amino acid residues containing a putative signal peptide of 19

227

amino acids. Using LBG as the substrate, PMan5A showed maximal activities at pH

228

5.0 (Fig. 1A), and remained stable in the pH range of 2.08.0 (Fig. 1B). For thermal

229

properties, PMan5A had a Topt of 70 °C and retained more than 90% of the initial

230

activity after preincubation at 60 °C for 30 min (Figs. 1C and 1D). Owing to the high

231

amino acid sequence identity of 73% between PMan5A and TlMan5A (Fig. S1), close

232

homolog TlMan5A had a higher Topt of 90 °C and retained high stability (no activity

233

loss at 75 °C for 30 min; Figs. 1C and 1D).20 Therefore, PMan5A and TlMan5A with

234

greatly different thermostabilities but high sequence similarity would be excellent

235

materials for studying the thermostable mechanism of GH5 -mannanases. 11



236

Rational design and thermal properties of chimeric mutants. To gain further

237

insight into the thermophilic mechanism of GH5 -mannanase, PMan5A and

238

TlMan5A20 were each divided into four domains based on multiple alignment analysis

239

of the primary sequences and secondary structures (Fig. S1), namely, Ala20-Ser118,

240

Tyr119-Gly195, Ile196-Gly289, and Arg290-Ala435 of PMan5A, and Val19-Thr114,

241

Tyr115-Asp191, Ile192-Asn285, and Arg286-Gln431 of TlMan5A (Fig. 2A). Four

242

chimeric proteins, M1–M4, were then obtained by swapping the sequence regions, as

243

shown in Fig. 2A. The thermostability of the chimeric enzymes was preliminarily

244

evaluated according to their Tm values.4 All chimeras had increased Tm values (Table

245

1). Among them, M1 showed a significant Tm increase of 7.7 °C, which was similar to

246

that of M2 and M3, while the Tm of M4 was 13.8 °C higher. This result suggested that

247

both the N- and C-termini of TlMan5A were critical for its thermostability, with their

248

introduction proving advantageous for improving the thermostability of PMan5A.

249

The N-terminal region of chimera M1 was then divided into four subdomains

250

(N1–N4), including the carbohydrate-binding module (CBM1), linker region,

251

antiparallel -sheet at the bottom of the barrel, and a -strand module (Fig. 2B). To

252

identify the most crucial submodule(s) for thermostability around the N-terminus,

253

three truncated enzymes, M1-ΔN1, M1-ΔN1N2, and M1-ΔN1N2N3, were

254

constructed. Compared to chimera M1, M1-ΔN1 and M1-ΔN1N2 showed similar Tm

255

values, while M1-ΔN1N2N3 was disabled without the antiparallel -sheet region of

256

the TIM barrel bottom (Table 1). These results suggested that the antiparallel -sheet

257

at the barrel bottom was necessary for protein folding. To investigate whether the 12


Page 12 of 45

Page 13 of 45


258

antiparallel -sheet contributed to thermostability, the N3 module of TlMan5A was

259

used to replace the corresponding region in PMan5A to give mutant enzyme PMan5A

260

(ΔN3) (Fig. 2B). DSC analysis indicated that the Tm value of mutant PMan5A (ΔN3)

261

was close to that of chimera M1 (Table 1). Therefore, the antiparallel -sheet at the

262

barrel bottom was inferred to be the focus of our in-depth study.

263

Considering the roles of terminal loops in thermostability,1,4 loop region

264

Glu368-Ala436 around the PMan5A C-terminus was also selected for further study.

265

Three loops, named C1, C2, and C3, were substituted with those of TlMan5A (Fig.

266

2C). Chimera M1-C2 had a similar Tm value to that of M1, while chimeras M1-C1

267

and M1-C3 had increased Tm values (1.8 and 5.8 °C higher, respectively; Table 1). In

268

particular, mutant M1-C3 exhibited a similar Tm value to TlMan5A (75.3 and

269

75.6 °C). Therefore, some key residues on the C-terminal loops (C1 and C3) were

270

inferred to be crucial for the thermostability of GH5 -mannanases.

271

Identification of critical sites. To determine whether and how residues in three

272

known modules (N3, C1, and C3) of PMan5A contributed to the thermal stability, the

273

multiple sequence alignment of 50 GH5 -mannanases from different organisms was

274

performed and the types of amino acid residues were counted. The results indicated

275

that PMan5A was distinct from the other counterparts at eleven sites in the selected

276

modules (Fig. 3A, Fig. S2), namely, Ala99, Ser100, Thr102, Val105, His112, Phe113,

277

Leu375, Ala408, Glu411, Ser413, and Phe415. The corresponding sequences of

278

PMan5A and TlMan5A were then aligned to identify the mutation sites (Fig. 3B).

279

Through site-directed mutagenesis, six amino acids at the antiparallel -sheet of the 13



280

barrel bottom of PMan5A were substituted with those of TlMan5A, namely, A99K,

281

S100N, T102L, V105T, H112Y, and F113Y. As shown in Table 2, double mutant

282

H112Y/F113Y had increased thermostability, with a Tm increase of 7.6 °C, which was

283

the same as that of chimera M1 (69.5°C). Using chimera M1 as the template, five

284

mutants in C-terminal regions C1 and C3, namely, M1-L375H, M1-A408P,

285

M1-E411G, M1-S413T, and M1-F415Y, were constructed and characterized. These

286

mutants showed Tm changes of 0.5 to +4.5 °C compared with that of chimera M1

287

(Table 2), with M1-L375H and M1-A408P showing more obvious changes. The

288

results suggested that the residues at positions 112, 113, 375, and 408 made a major

289

contribution to the thermostability of PMan5A.

290

Thermal properties of residue-substituted mutants. Single and multiple

291

mutations at H112, F113, L375, and A408 were completed using site-directed

292

mutagenesis. Single-site mutants H112Y and A408P showed improved activities at

293

high temperature (Fig. 4A), with maximal activities at 80 and 75 °C, respectively. The

294

Topt values were increased by 10 and 5 °C, respectively, compared with that of

295

wild-type PMan5A (70 °C). Double-site mutant H112Y/A408P had a Topt of 85 °C,

296

suggesting the occurrence of certain additive effects. Although mutants F113Y and

297

L375H had similar Topt values to that of the wild type, they showed improved

298

activities at high temperatures. Triple- and quadruple-site mutants

299

H112Y/L375H/A408P and H112Y/F113Y/L375H/A408P retained higher activities at

300

90 °C than the double mutant (H112Y/A408P) (approximately 38% and 50% vs.

301

approximately 28%). These results indicated that the four selected positions made 14


Page 14 of 45

Page 15 of 45

302 303


outstanding contributions to the thermostability. The thermal tolerance (T50) was investigated to determine the kinetic stability of

304

these mutant enzymes. Wild-type PMan5A had a T50 value of 66 °C, which was much

305

lower than those of the single and multiple-site mutants (Fig. 3B). For single mutants,

306

the effect on thermal tolerance followed the order H112Y>A408>L375H>F113Y.

307

After preincubation for 30 min at 80 °C, no activity was detected in wild-type

308

PMan5A and the four single-site mutants, while the combined mutants H112Y/A408,

309

H112Y/L375H/A408, and H112Y/F113Y/L375H/A408 retained 24%, 38%, and 53%

310

of their activities, respectively.

311

The half-life (t1/2) was also determined to assay the enzyme stability in the

312

absence of substrate. After preincubation at 75 °C for different durations, the t1/2 of

313

PMan5A was found to be 2 min, while the four single-site mutants, H112Y, F113Y,

314

L375H, and A408P, had t1/2 values of approximately 10, 2, 4, and 5 min, respectively

315

(Table 3). Furthermore, the t1/2 of H112Y/A408P reached 120 min at 75 °C, while

316

quadruple-site mutant H112Y/F113Y/L375H/A408P showed the best thermal

317

stability, with t1/2 values of 180 and 30 min at 75 and 80 °C, respectively. These

318

results were consistent with the corresponding Tm and Topt values (75.5 °C and 85 °C).

319

Therefore, we inferred that mutants H112Y, F113Y, L375H, and A408P had additive

320

effects on the thermostability.

321

Specific activity and kinetic parameters of PMan5A mutants. Under optimal

322

conditions (7085 °C and pH 5.0), the specific activities and kinetic parameters of

323

PMan5A and its mutants were analyzed using LBG as the substrate (Table 4). 15



324

Compared with PMan5A, all mutants except L375H showed improved specific

325

activities (1.21.7-fold). Furthermore, the substitution of specific residues greatly

326

influenced the kinetic parameters, including slightly decreased substrate binding

327

affinities (elevated Km values) and increased turnover numbers (elevated kcat values).

328

Therefore, the catalytic efficiency (kcat/Km) of all mutants except A408P and F113Y

329

was increased 1.11.6-fold. Notably, this effect was additive, with the quadruple

330

mutant H112Y/F113Y/L375H/A408P exhibiting the best catalytic capability (highest

331

kcat/Km and specific activity), which was close to that of thermophilic parent

332

TlMan5A.

333

Reverse mutations on TlMan5A. To validate the effect of positions 112, 113,

334

375, and 408 on the thermal stability, reverse mutations were performed on the GH5

335

-mannanase TlMan5A. The corresponding Tyr108, Tyr109, His371, and Pro406

336

residues of TlMan5A were substituted with histidine, phenylalanine, lysine, and

337

alanine, respectively, to generate three mutants, namely, TlMan5A_Y108H,

338

TlMan5A_Y108H/H371L/P406A, and TlMan5A_Y108H/Y109F/H371L/P406A. All

339

enzymes were successfully expressed in P. pastoris GS115.

340

Using LBG as the substrate, TlMan5A and its variants were optimally active at

341

approximately pH 4.5 (Fig. S3C). The result indicated that mutations had almost no

342

effect on the pH–activity profiles of TlMan5A, which was consistent with the forward

343

mutations of PMan5A. However, a significant decline in the thermophilic properties

344

was detected in the TlMan5A mutants. Wild-type (TlMan5A), TlMan5A_Y108H,

345

TlMan5A_Y108H/H371L/P406A, and TlMan5A_Y108H/Y109F/H371L/P406A 16


Page 16 of 45

Page 17 of 45


346

showed optimal activities at pH 4.5 and temperatures of 90, 80, 70 and 70 °C,

347

respectively (Fig. S3A). These four enzymes retained 100, 90, 17, and 17% of their

348

initial activity after incubation for 30 min at 70 °C, respectively (Fig. S3B). These

349

results suggested that four residues, valine, valine, histidine, and proline, at the 112,

350

113, 375, and 408 positions did contribute to the thermal stability of PMan5A in GH5

351

-mannanase.

352

MD simulation. MD simulations of PMan5A and its mutant

353

H112Y/F113Y/L375H/A408P at 300 K were conducted at 50 ns. As shown in Fig.

354

5A, residues His112 and Phe113 were located near the antiparallel -sheet at the

355

barrel bottom of PMan5A. The side chain of residue His112 was confined to the

356

center of the hydrophobic area formed by residues Ala114, Ile387, Ala391, and

357

Val429 (Fig. 5C). After mutation, the overall orientations of the residues at position

358

112 in the two systems were similar (Fig. 5D). However, analyzing the hydrophobic

359

core networks of the two systems provided detailed information about conformational

360

differences. The side chain of residue Ile432 became closer to the Tyr112 residue,

361

with a distance of 4.1 Å (vs. 11.2 Å in PMan5A) between the CB atom and centroid

362

of Tyr112 (Fig. 5B). This shorter distance in the H112Y/F113Y/L375H/A408P

363

mutant might enhance the hydrophobic interactions.

364

The side chain of Phe113 pointed to the barrel bottom of PMan5A and was

365

grappled by hydrophobic interaction with Ile106 (Fig. 5C). Meanwhile, two stable salt

366

bridges, Lys109-Asp107 and Lys143-Asp107, were identified in the antiparallel

367

-sheet at the barrel bottom, which had occupancy rates of 80.47% and 75.78% in the 17



368

trajectories, respectively. However, the conformation of the representative structures

369

changed (Fig. 5D), with the CG atom of Lys143 and Tyr113 having an occupancy rate

370

of 65.69%, while Lys197-Asp107, the most stable salt bridge formed in the mutant

371

H112Y/F113Y/L375H/A408P, had an occupancy rate of 76.43%.

372

Asp411 has reportedly been located at loop 8, the key region related to substrate

373

specificity,36 where it specifically binds to the galactosyl unit in subsite 1 (Fig. 6A).

374

To determine whether these structures determined the changes in thermostability,

375

root-mean-square fluctuations (RMSF) computed from MD simulations were plotted

376

for residues in the proximity of loop 8 for both PMan5A and its mutant

377

H112Y/F113Y/L375H/A408P. Generally speaking, RMSF measures the amplitude of

378

atom motion during a simulation, with a higher RMSF value reflecting a higher

379

flexibility. As shown in Fig. 6B, loop 8 was more flexible in the wild type than in

380

mutant H112Y/F113Y/L375H/A408P, reinforcing that this portion was more rigid in

381

the mutant, probably accounting for the improved thermostability. Residues Leu375

382

and Ala408, located on loops 7 and 8, respectively, were found to form hydrophobic

383

interactions with residues Trp36 and Tyr328 (Fig. 6C). Meanwhile, the OH atom of

384

Tyr328 and the OD1 atom of Asp326 formed a hydrogen bond, with an occupancy

385

rate of 51.23%. Furthermore, the side chain of Tyr328 was flipped by approximately

386

45° (Fig. 6D), which brought the aromatic nucleus closer to loop 6, while residue

387

His375 moved to interact with Ile330 and Leu340. These molecular interactions might

388

explain the improved thermophilic properties of quadruple mutant

389

H112Y/F113Y/L375H/A408P. 18


Page 18 of 45

Page 19 of 45

390


DISCUSSION

391

GH5 endo--mannanase is an important catalyst for the deconstruction of

392

hemicellulose in softwoods.37 Thermostable mannanases, which have intrinsic

393

stability and activity at high temperatures, have biotechnological advantages in

394

numerous bioprocesses.38,39 In past decades, much effort has been focused toward

395

understanding the molecular basis of -mannanase thermostability. For example, the

396

Ca2+ binding sites located on loops 6 and 7 in an actinomycete mannanase (StMan)

397

were found to be responsible for the enhanced thermal stability obtained by forming

398

hydrogen bonds between residues Ser247 and Asp279.40 After removing disulfide

399

linkages, the B. subtilis z-2 mannanase mutant (BcMan) showed weakened thermal

400

properties.41 CBM also plays a role in the thermostability of endo--mannanases,

401

although function in thermostability remains unknown.16,42 In the present study, we

402

combined module substitution, region truncation, and site-directed mutagenesis to

403

identify the key structural elements (terminal modules, loops, and key residues) near

404

the N- and C- termini that were related to the thermostability of GH5 -mannanase

405

PMan5A.

406

During thermal denaturation, unfolding starts from the loose and diverse N- and

407

C-termini.1 Therefore, these two unstable regions might affect the protein thermal

408

properties by modifying the modules or structures.23,43 Among the (/)8-barrel

409

enzymes, the “stability face” at the N-terminus of the sequence formed the bottom

410

capping the -barrel at the opposite end of the “catalytic face”, which consisted of

411

residues that could maintain the protein stability.13 The significant increase (~7.6 °C) 19



412

in apparent Tm of the mutant PMan5A (ΔN3) might be attributed to the antiparallel

413

-sheet at the bottom. Similarly, truncation of the N-terminal 34 residues that form a

414

-sheet structure caused a significant decreased in the stability of -endoglucanase

415

from P. horikoshii (EGPh).23 Therefore, the “stability face” of canonical TIM barrels,

416

such as PMan5A, might function through the structural element of the N-terminus,

417

especially the extra short -strands of the -barrel. Furthermore, our modular

418

truncation analysis showed that deleting the antiparallel -sheet from the TIM barrel

419

bottom (M1-ΔN1ΔN2ΔN3) led to enzyme inactivation, while deleting CBM and the

420

linker region had no effect on the thermostability and enzyme activity (Table 1).

421

These results suggested that the domain at the bottom of TIM barrel was necessary for

422

protein folding.

423

Amino acid interactions play a fundamental role in protein thermostability.44,45

424

MD simulations suggested that mutations H112Y and F113Y had a stronger

425

hydrophobic effect (Ile432-Tyr112) and more salt bridges (Lys143-Tyr113 and

426

Lys197-Asp107) in the inner wall of the -barrel. Furthermore, molecular interaction

427

analysis showed that the interactions all occurred between the domain of the TIM

428

barrel bottom and the barrel core structure. Therefore, the domain at the bottom of the

429

TIM barrel was indeed important for achieving enhanced global conformational

430

rigidity in the GH5 β-mannanases. Further studies showed that His112 and Phe113

431

located near the domain at the bottom of the (/)8-barrel (Fig. 5A) played a critical

432

role in the thermoresistance and catalytic performance of GH5 -mannanases. To our

433

knowledge, the present study is the first to elucidate the roles of the peripheral 20


Page 20 of 45

Page 21 of 45


434

domain, such as the role of the two antiparallel -stands at the bottom of TIM barrel

435

in the thermostability of (/)8-barrel enzymes, through experiments and MD

436

simulations.

437

Loops are essential structural elements and effect the protein flexibility/rigidity

438

patterns and corresponding thermostabilization.46 For example, shortening the loop of

439

the acylphosphatase (AcP) increased the conformational entropy and promoted

440

thermal stabilization,47 while substituting loop DE enhanced the thermal stability of a

441

mesophilic GH5 -mannanase.30 Our studies are consistent with previous results,

442

showing that substituting loops 7 and 8 in chimeras M1-C1 and M1-C3 increased the

443

Tm value by ~1.8 and ~5.8 °C, respectively. These loops of GH5 mannanases from

444

actinomycete have been confirmed to participate in linear substrate recognition, which

445

is related to increased catalytic efficiency.48 Furthermore, proline on loops can

446

strengthen the adjacent structures and improve protein rigidity. For example, the

447

introduction of proline in -amylases increases the rigidity of the C-terminal region

448

outside of the barrel core.25 Therefore, mutation A408P on loop 8 enhanced the

449

protein rigidity and improved the Tm and half-life by ~5.0 °C and 1.5-fold,

450

respectively. Interestingly, the effect of single-site mutagenesis on protein

451

thermostabilization was independent and additive, with a strong synergy observed in

452

quadruple mutant H112Y/F113Y/L375H/A408P (Table 3).

453

In conclusion, we have developed a systematic protein engineering strategy to

454

explore the molecular basis of the thermostability of GH5 -mannanases. Four key

455

residues, Y112, Y113, H375, and P408, were identified to play favorable roles in 21



456

mediating the thermostability of GH5 -mannanase PMan5A, in the order

457

H112Y>A408P>L375H>F113Y, as observed for the Tm, T50, and t1/2 values. The

458

combined multiple loci mutants, compared with the corresponding single-site

459

mutants, showed a strong synergistic effect on the thermal stability, while quadruple

460

mutant H112Y/F113Y/L375H/A408P, which showed the greatest shift in thermal

461

resistance to high temperatures, had almost the same level of thermostability as

462

TlMan5A. MD simulation analysis showed that the domain at the barrel bottom,

463

outside of barrel core, was critical for structure stabilization, and that residues on

464

loops 7 and 8 of the C-terminus were related to both thermostability and catalytic

465

efficiency. This study not only elucidates the key module and residues related to

466

enzyme thermostability, but also suggests strategies for improving GH5 -mannanase.

467 468

22


Page 22 of 45

Page 23 of 45

469 470


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

471

China (Grant No. 31601976), the National Special Program for GMO Development of

472

China (Grant No. 2016ZX08003-002) and the China Modern Agriculture Research

473

System (Grant No. CARS-41).

474

Supporting Information

475

Table S1. The primers used in this study.

476

Fig. S1. Multiple sequence alignment of PMan5A and TlMan5A (GenBank:

477

KJ607175) with the secondary structure elements of ManBK (PDB ID: 3WH9) as the

478

template. Fig. S2. Multiple sequence alignments of 50 fungal -mannanase of GH5.

479

Three modules of N3, Loop 7 and 8 are labeled by braces. Fig. S3. Enzymatic

480

properties of the wild type TlMan5A and their variants. (A) Temperature-activity

481

profiles of TlMan5A and its variants tested at the optimal pH of each enzyme in the

482

temperature range of 30–95 °C for 10 min. (B) Thermostability of TlMan5A and its

483

variants investigated after 30-min incubation without substrate at indicated

484

temperatures and pH 4.5. (C) pH-activity profiles of TlMan5A and its variants tested

485

at the optimal temperature of each enzyme (70-90 °C) over the pH range of 3.0–7.0

486

for 10 min.

487 488 489 490 23



491

REFERENCES

492

(1) Vieille, C.; Zeikus, G.J. Hyperthermophilic enzymes: sources, uses, and

493

molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 2001, 65

494

(1), 1–43.

495

(2) Broom, A.; Jacobi, Z.; Trainor, K.; Meiering, E.M. Computational tools help

496

improve protein stability but with a solubility tradeoff. J. Biol. Chem. 2017,

497

292(35), 14349–14361.

498

(3) Rigoldi, F.; Donini, S.; Redaelli, A.; Parisini, E.; Gautieri, A. Engineering of

499

thermostable enzymes for industrial applications. APL Bioengineer. 2018, 2(1),

500

011501.

501 502 503

(4) Pucci, F.; Rooman, M. Physical and molecular bases of protein thermal stability and cold adaptation. Curr. Opin. Struct. Biol. 2017, 42, 117–128. (5) Xie, Y.; An, J.; Yang, G.; Wu, G.; Zhang, Y.; Cui, L.; Feng, Y. Enhanced

504

enzyme kinetic stability by increasing rigidity within the active site. J. Biol.

505

Chem. 2014, M113, 536045.

506

(6) Hilge, M.; Gloor, S.M.; Rypniewski, W.; Sauer, O.; Heightman, T.D.;

507

Zimmermann, W.; Piontek, K. High-resolution native and complex structures of

508

thermostable β-mannanase from Thermomonospora fusca-substrate specificity in

509

glycosyl hydrolase family 5. Structure 1998, 6(11), 1433–1444.

510 511 512

(7) Matsui, I.; Harata, K. Implication for buried polar contacts and ion pairs in hyperthermostable enzymes. FEBS J. 2007, 274(16), 4012–4022. (8) Davies, G.J.; Gamblin, S.J.; Littlechild, J.A.; Watson, H.C. The structure of a 24


Page 24 of 45

Page 25 of 45


513

thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic

514

equivalent. Proteins 1993, 15(3), 283–289.

515 516 517

(9) Kumar, S.; Tsai, C.J.; Nussinov, R. Factors enhancing protein thermostability. Protein Eng. 2000, 13(3), 179–191. (10) Malgas, S.; van Dyk, J.S.; Pletschke, B.I. A review of the enzymatic hydrolysis

518

of mannans and synergistic interactions between β-mannanase, β-mannosidase

519

and α-galactosidase. World J. Microbiol. Biotechnol. 2015, 31(8), 11671175.

520

(11) Ghosh, A.; Luís, A.S.; Brás, J.L.A.; Fontes, C.M.; Goyal, A. Thermostable

521

recombinant is of β-(1→4)-mannanase from C. thermocellum: biochemical

522

characterization and manno-oligosaccharides production. J. Agric. Food. Chem.

523

2013, 61(50), 1233312344.

524

(12) Tailford, L.E., Ducros, V.M.A., Flint, J.E., Roberts, S.M., Morland, C., Zechel,

525

D.L., Smith, N., Bjørnvad, M.E., Borchert, T.V., Wilson, K.S., Davies, G.J.,

526

Gilbert, H.J. Understanding how diverse β-mannanases recognize heterogeneous

527

substrates. Biochemistry-US 2009, 48(29), 70097018.

528

(13) Couturier, M.; Roussel, A.; Rosengren, A.; Leone, P.; Stålbrand, H.; Berrin, J.G.

529

Structural and biochemical analyses of glycoside hydrolase families 5 and 26

530

β-(1,4)-mannanases from Podospora anserina reveal differences upon

531

manno-oligosaccharide catalysis. J. Biol. Chem. 2013, 288(20), 1462414635.

532

(14) Sterner, R.; Höcker, B. Catalytic versatility, stability, and evolution of the

533

(βα)8-barrel enzyme fold. Chem. Rev. 2005, 105(11), 4038–4055.

534

(15) Sharma, K.; Dhillon, A.; Goyal. A. Insights into structure and reaction 25



535 536

mechanism of β-mannanases. Curr. Protein Pept. Sc. 2016, 19, 34–47. (16) Sunna, A. Modular organisation and functional analysis of dissected modular

537

β-mannanase CsMan26 from Caldic ellulosiruptor Rt8B.4. Appl. Microbiol.

538

Biotechnol. 2010, 86(1), 189–200.

539

(17) Pham, T.A.; Berrin, J.G.; Record, E.; To, K.A.; Sigoillot, J.C. Hydrolysis of

540

softwood by Aspergillus mannanase: role of a carbohydrate-binding module. J.

541

Biotechnol. 2010, 148(4), 163–170.

542

(18) Boraston, A.B.; Bolam, D.N.; Gilbert, H.J.; Davies, G.J. Carbohydrate-binding

543

modules: Fine-tuning polysaccharide recognition. Biochem. J. 2004, 382(3),

544

769–781.

545

(19) Hervé, C.; Rogowski, A.; Blake, A.W.; Marcus, S.E.; Gilbert, H.J.; Knox, J.P.

546

Carbohydrate-binding modules promote the enzymatic deconstruction of intact

547

plant cell walls by targeting and proximity effects. Proc. Natl. Acad. Sci. 2010,

548

107(34), 15293–15298.

549

(20) Wang, C.; Luo, H.; Niu, C.; Shi, P.; Huang, H.; Meng, K.; Bai, Y.; Wang, K.;

550

Hua, H.; Yao, B. Biochemical characterization of a thermophilic β-mannanase

551

from Talaromyces leycettanus JCM12802 with high specific activity. Appl.

552

Microbiol. Biotechnol. 2015, 99(3), 1217–1228.

553

(21) Tang, C.D.; Li, J.F.; Wei, X.H.; Min, R.; Gao, S.J.; Wang, J.Q.; Yin, X.; Wu,

554

M.C. Fusing a carbohydrate-binding module into the Aspergillus usamii

555

β-mannanase to improve its thermostability and cellulose-binding capacity by in

556

silico design. PLoS One. 2013, 8: e64766. 26


Page 26 of 45

Page 27 of 45

557


(22) Lu, H.; Luo, H.; Shi, P.; Huang, H.; Meng, K.; Yang, P.; Yao, B. A novel

558

thermophilic endo-β-1,4-mannanase from Aspergillus nidulans XZ3: functional

559

roles of carbohydrate-binding module and Thr/Ser-rich linker region. Appl.

560


561

(23) Yang, T.C.; Legault, S.; Kayiranga, E.A.; Kumaran, J.; Ishikawa, K.; Sung, W.L.

562

The N-terminal β-sheet of the hyperthermophilic endoglucanase from

563

Pyrococcus horikoshii is critical for thermostability. Appl. Environ. Microbiol.

564

2012, 78(9), 3059–3067.

565

(24) Uraji, M.; Tamura, H.; Mizohata, E.; Arima, J.; Wan, K.; Ogawa, K.I.; Noue, T.;

566

Hatanaka, T. Loop of Streptomyces feruloyl esterase plays an important role in

567

the enzyme's catalyzing the release of ferulic acid from biomass. Appl. Environ.

568

Microbiol. 2018, 84(3), e02300.

569

(25) Yang, G.; Yao, H.; Mozzicafreddo, M.; Ballarini, P.; Pucciarelli, S.; Miceli, C.

570

Rational engineering of a cold-adapted α-amylase from the Antarctic ciliate

571

Euplotes focardii for simultaneous improvement of thermostability and catalytic

572

activity. Appl. Environ. Microbiol. 2017, 83(13), e00449.

573

(26) Goedegebuur, F.; Dankmeyer, L.; Gualfetti, P.; Karkehabadi, S.; Hansson, H.;

574

Jana, S.; Huynh, V.; Kelemen, B.R.; Kruithof, P.; Larenas, E.A.; Teunissen, P.J.;

575

Ståhlberg, X.J.; Payne, C.M.; Mitchinson, C.; Sandgren, M. Improving the

576

thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed

577

evolution. J. Biol. Chem. 2017, 292(42), 1741817430.

578

(27) Li, J.M.; Nie, S.P. The functional and nutritional aspects of hydrocolloids in 27



579 580

foods. Food Hydrocolloid. 2016, 53, 4661. (28) Nadaroglu, H.; Adiguzel, A.; Adiguzel, G. Purification and characterisation of

581

β-mannanase from Lactobacillus plantarum (M24) and its applications in some

582

fruit juices. Int. J. Food Sci. Technol. 2015, 50(5), 1158–1165.

583

(29) Lee, J.J.; Seo, J.; Jung, J.K.; Lee, J.; Lee, J.H.; Seo, S. Effects of β-mannanase

584

supplementation on growth performance, nutrient digestibility, and nitrogen

585

utilization of Korean native goat (Capra hircus coreanae). Livest. Sci. 2014, 169,

586

83–87.

587

(30) Dong, Y.H.; Li, J.F.; Yin, X.; Wang, C.J.; Tang, S.H.; Wu, M.C. Replacing a

588

piece of loop-structure in the substrate-binding groove of Aspergillus usamii

589

β-mannanase, AuMan5A, to improve its enzymatic properties by rational design.

590

Appl. Microbiol. Biotechnol. 2016, 100(9), 3989–3998.

591 592 593 594 595

(31) Srivastava, P.K.; Kapoor, M. Production, properties, and applications of endo-β-mannanases. Biotechnol. Adv. 2017, 35(1), 1–19. (32) Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31(3), 426–428. (33) Huag, J.W.; Chen, C.C.; Huang, C.H.; Huang, T.Y.; Wu, T.H.; Cheng, Y.S.;

596

Guo, R.T. Improving the specific activity of β-mannanase from Aspergillus niger

597

BK01 by structure-based rational design. BBA-Proteins Proteome. 2014,

598

1844(3), 663–669.

599

(34) Kirschner, K.N.; Yongye, A.B.; Tschampel, S.M.; González-Outeiriño, J.;

600

Daniels, C.R.; Foley, B.L.; Woods, R.J. GLYCAM06: a generalizable 28


Page 28 of 45

Page 29 of 45


601

biomolecular force field. Carbohydrates. J. Comput. Chem. 2008, 29(4), 622–

602

655.

603 604 605

(35) 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(12), 10089–10092. (36) Ryckaert, J.P.; Ciccotti, G.; Berendsen, H.J. Numerical integration of the

606

cartesian equations of motion of a system with constraints: molecular dynamics

607

of n-alkanes. J. Comput. Phys. 1977, 23(3), 327–341.

608

(37) Chauhan, P.S.; Puri, N.; Sharma, P.; Gupta, N. Mannanases: microbial sources,

609

production, properties and potential biotechnological applications. Appl.

610


611

(38) Prima, A.; Hara, K.Y.; Djohan, A.C.; Kashiwagi, N.; Kahar, P.; Ishii, J.;

612

Nakayama, H.; Okazaki, F.; Prasetya, B.; Kondo, A. Glutathione production from

613

mannan-based bioresource by mannanase/mannosidase expressing

614

Saccharomyces cerevisiae. Bioresour. Technol. 2017, 245, 1400–1406.

615

(39) Nguyen, H.M.; Mathiesen, G.; Stelzer, E.M.; Pham, M.L.; Kuczkowska, K.;

616

Mackenzie, A.; Agger, J.W.; Eijsink, V.G.; Yamabhai, M.; Peterbauer, C.K.;

617

Haltrich, D.; Nguyen, T.H. Display of a β-mannanase and a chitosanase on the

618

cell surface of Lactobacillus plantarum towards the development of whole-cell

619

biocatalysts. Microb. Cell Fact. 2016, 15(1), 169.

620

(40) Kumagai, Y.; Uraji, M.; Wan, K.; Okuyama, M.; Kimura, A.; Hatanaka, T.

621

Molecular insights into the mechanism of thermal stability of actinomycete

622

mannanase. FEBS Lett. 2016, 590(17), 28622869. 29



623

(41) Yan, X.X.; An, X.M.; Gui, L.L.; Liang, D.C. From structure to function: insights

624

into the catalytic substrate specificity and thermostability displayed by Bacillus

625

subtilis mannanase BCman. J. Mol. Biol. 2008, 379(3), 535544.

626

(42) dos Santos, C.R.; Paiva, J.H.; Meza, A.N.; Cota, J.; Alvarez, T.M.; Ruller, R.;

627

Prade, R.A.; Squina, F.M.; Murakami, M.T. Molecular insights into substrate

628

specificity and thermal stability of a bacterial GH5-CBM27

629

endo-1,4-β-D-mannanase. J. Struct. Biol. 2012, 177(2), 469–476.

630

(43) Srivastava, P.; Appu Rao, A.G.; Kapoor, M. Structural insights into the thermal

631

stability of endo-mannanase belonging to family 26 from Bacillus sp. CFR1601.

632

FASEB J. 2014, 28(suppl. 1), 580.2.

633

(44) Saelensminde, G.; Halskau, O.J.; Jonassen, I. Amino acid contacts in proteins

634

adapted to different temperatures: hydrophobic interactions and surface charges

635

play a key role. Extremophiles 2009, 13(1), 11–20.

636

(45) Wolfenden, R.; Lewis, C.A.; Yuan, Y.; Carter, C.W. Temperature dependence of

637

amino acid hydrophobicities. Proc. Natl. Acad. Sci. 2015, 112(24), 7484–7488.

638

(46) Gavrilov, Y.; Dagan, S.; Levy, Y. Shortening a loop can increase protein native

639 640

state entropy. Proteins 2015, 83(12), 2137–2146. (47) Dagan, S.; Hagai, T.; Gavrilov, Y.; Kapon, R.; Levy, Y.; Reich, Z. Stabilization

641

of a protein conferred by an increase in folded state entropy. Proc. Natl. Acad.

642

Sci. 2013, 110(26), 10628–10633.

643 644

(48) Kumagai, Y.; Yamashita, K.; Tagami, T.; Uraji, M.; Wan, K.; Okuyama, M.; Hatanaka, T. The loop structure of Actinomycete glycoside hydrolase family 5 30


Page 30 of 45

Page 31 of 45

645


mannanases governs substrate recognition. FEBS J. 2015, 282(20), 4001–4014.

31



647

FIGURE CAPTIONS

648

Fig 1 Enzymatic properties of PMan5A and TlMan5A. (A) pH effects on the enzyme

649

activities tested at optimal temperature (70 and 90°C) over the pH range of 3.0–7.0

650

for 10 min. (B) pH stability determined at optimal pH and temperature for 10 min

651

after 1-h incubation at indicated pH and 37°C without substrate. (C) Temperature

652

effects on the enzyme activities tested at indicated temperatures and pH 4.5

653

(TlMan5A) or 5.0 (PMan5A) for 10 min. (D) Thermostability investigated after

654

30-min incubation without substrate at indicated temperatures and pH 4.5 (TlMan5A)

655

or 5.0 (PMan5A).

656

Fig 2 Schematic construction of the chimeric mutants. The sequences of TlMan5A

657

and PMan5A are marked in yellow and blue, respectively. aa, amino acids. (A)

658

Module substitution. (B) Truncation of the N-terminal sequences with chimera M1 as

659

the template. (C) Substitution of the C-terminal sequences with chimera M1 as the

660

template.

661

Fig 3 Identification of distinct residues of PMan5A. (A) Distinct residues derived

662

from the multiple sequence alignment of 50 GH5 β-mannanases. (B) Distinct residues

663

derived from the sequence alignment with TlMan5A.

664

Fig 4 Temperature effects on the hydrolytic and stable capabilities of PMan5A and its

665

mutants. (A) Specific activities determined at indicated temperatures and pH 5.0 for

666

10 min. (B) Thermostability investigated after 30-min incubation without substrate at

667

indicated temperatures.

668

Fig 5 Conformation analysis of residue 112 and 113 in the MD trajectory of the 32


Page 32 of 45

Page 33 of 45


669

enzymes. (A) The modeled structure of PMan5A viewed from the barrel bottom. The

670

antiparallel β-sheet of the barrel bottom is depicted as green. (B) The distance

671

between the CB atom of residue Ile432 and the centroid of Tyr112. The side chains of

672

residue 112 and 113 participating in the detailed interactions in PMan5A (C) and

673

mutant H112Y/F113Y/L375H/A408P (D).

674

Fig 6 Conformation analysis of residue 375 and 408 in the MD trajectory of the

675

enzymes. (A) The modeled structure of PMan5A viewed from the catalytic pocket.

676

The loop 8 is depicted as blue. (B) Root-mean-square fluctuations (RMSF) computed

677

from MD simulations for PMan5A and mutant H112Y/F113Y/L375H/A408P. The

678

side chains of residue 328 and 408 participating in the detailed interactions in

679

PMan5A (C) and mutant H112Y/F113Y/L375H/A408P (D).

680 681 682 683 684 685 686

33



Page 34 of 45

TABLE 1 Tm values of the wild-type and chimeric β-mannanases.a Enzyme

Tm (°C)

PMan5A

61.8 ± 0.0

PMan5A(ΔN3)

69.3 ± 0.2

M1

ΔTm b (°C) Enzyme

Tm (°C)

ΔTm (°C)

M1-ΔN1

69.5 ± 0.3

+7.7

+7.6

M1-ΔN1N2

69.3 ± 0.1

+7.6

69.5 ± 0.1

+7.7

M1-ΔN1N2N3

ND

ND

M2

69.3 ± 0.9

+7.5

M1-C1

71.3 ± 0.2

+9.5

M3

69.8 ± 0.5

+8.0

M1-C2

69.5 ± 0.1

+7.7

M4

75.6 ± 0.1

+13.8

M1-C3

75.3 ± 0.1

+13.6

aT

m values

are shown as means ± standard deviations (n = 3); b ΔTm is Tm value minus

the Tm value of PMan5A (wild-type). ND, no activity detected.

34


Page 35 of 45


TABLE 2 Tm values of the parent and mutant β-mannanases. a Enzymes

Tm (°C)

Pman5A

61.8 ± 0.0

H112Y/F113Y

69.4 ± 0.1

A99K/S100N

ΔTm b (°C)

Enzymes

Tm (°C)

ΔTm c(°C)

M1

69.5 ± 0.1

+7.6

M1-L375H

71.0 ± 0.0

+1.6

62.7 ± 0.4

+0.9

M1-A408P

74.0 ± 0.3

+4.5

T102L

62.5 ± 0.2

+0.7

M1-E411G

69.2 ± 0.1

0.3

V105T

61.7 ± 0.1

+0.0

M1-S413T

69.7 ± 0.1

+0.2

M1-F415Y

69.6±0.1

+0.2

aT

m

values are shown as means ± standard deviations (n = 3). b ΔTm is Tm value minus

the Tm value of PMan5A (wild-type). c ΔTm is Tm value minus the Tm value of M1.

35



Page 36 of 45

TABLE 3 t1⁄2 and Tm values of the parent and mutant β-mannanases. t1/2 (min)

Tm a (°C)

ΔTm b (°C)

Enzymes 75 °C Pman5A

80 °C

2

ND

61.8 ± 0.0

H112Y

10

3

69.2 ± 0.0

+7.4

F113Y

2

ND

61.8 ± 0.1

+0.0

L375H

4

ND

63.5 ± 0.2

+1.7

A408P

5

2

66.8 ± 0.2

+5.0

H112Y/F113Y

17

3

69.4 ± 0.1

+7.6

H112Y/L375H

55

5

70.4 ± 0.0

+8.6

H112Y/A408P

120

14

71.9 ± 0.1

+10.1

L375H/A408P

5

3

67.4 ± 0.1

+5.6

H112Y/F113Y/L375H

47

5

69.7 ± 0.3

+7.9

H112Y/L375H/A408P

120

15

75.3 ± 0.1

+13.5

H112Y/F113/L375H/A408P

180

30

75.5 ± 0.1

+13.8

aT

m

values are shown as means ± standard deviations (n = 3). b ΔTm is Tm value minus

the Tm value of PMan5A (wild-type). ND, not determined.

36


Page 37 of 45


Table 4 Kinetic values of the parent and mutant β-mannanases with LBG as the substrate. Specific activity

Km

Vmax

kcat

kcat/Km

(U/mg)

(mg/mL)

(μmol/min·mg)

(/s)

(mL/s·mg)

PMan5A

1276 ± 19

0.51 ± 0.07

1115 ± 43

836 ± 32

1628 ± 144

H112Y

1537 ± 47

0.68 ± 0.12

1862 ± 154

1397 ± 116

2066 ± 213

F113Y

1612 ± 92

0.87 ± 0.04

2048 ± 59

1536 ± 44

1758 ± 98

L375H

1237 ± 43

0.87 ± 0.11

2489 ± 105

1867 ± 79

2155 ± 165

A408P

1609 ± 15

0.86 ± 0.07

1679 ± 45

1259 ± 33

1471 ± 101

H112Y/F113Y

1769 ± 54

0.70 ± 0.09

1941 ± 119

1456 ± 89

2067 ± 125

H112Y/L375H

1585 ± 19

0.68 ± 0.06

2046 ± 76

1535 ± 57

2263 ± 200

H112Y/A408P

1742 ± 75

0.81 ± 0.05

2261 ± 75

1696 ± 56

2081 ± 80

Enzymes

37



Page 38 of 45

L375H/A408P

1712 ± 23

0.73 ± 0.08

1977 ± 71

1483 ± 53

2039 ± 148

H112Y/F113Y/L375H

1921 ± 64

0.58 ± 0.05

1641 ± 30

1231 ± 23

2134 ± 158

H112Y/L375H/A408P

2202 ± 38

0.73 ± 0.03

2468 ± 66

1851 ± 50

2549 ± 51

H112Y/F113Y/L375H/A408P

2226 ± 178

0.67 ± 0.02

2225 ± 52

1669 ± 39

2485 ± 17

38


Page 39 of 45


FIG 1



FIG 2


Page 40 of 45

Page 41 of 45


FIG 3



FIG 4


Page 42 of 45

Page 43 of 45


FIG 5



FIG 6


Page 44 of 45

Page 45 of 45



Insight into the Thermophilic Mechanism of a Glycoside Hydrolase

Recommend Documents