Crystal structure and biochemical characterization of an

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Crystal structure and biochemical characterization of an aminopeptidase LapB from Legionella pneumophila NanNan Zhang, Shiyan Yin, Wei Zhang, Xiaojian Gong, Na Zhang, Kai Fang, and Honghua Ge J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02849 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 6, 2017

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Journal of Agricultural and Food Chemistry

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Crystal

structure

and

biochemical

characterization

of

an

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aminopeptidase LapB from Legionella pneumophila

3

Nannan Zhang, Shiyan Yin, Wei Zhang, Xiaojian Gong, Na Zhang, Kai Fang,

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Honghua Ge *

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Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, Anhui

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230601, China

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*Correspondence to: Honghua Ge, Institute of Health Sciences, School of Life

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Sciences, Anhui University, Hefei, Anhui 230601, China.

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Tel & Fax: +86-0551-63861773

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Aminopeptidases are a group of exopeptidases that catalyze the removal of a wide

25

range of N-terminal amino acid residues from peptides and proteins. They have many

26

important commercial applications in the food industry. We determined the crystal

27

structure of an aminopeptidase LapB from Legionella pneumophila. The overall

28

structure reveals that the N-terminal protease-associated (PA) domain presents a new

29

fold and shields the active site cavity of the conserved C-terminal peptidase domain.

30

The steady-state kinetic analysis of LapB and the PA domain deletion mutant indicate

31

that the PA domain inhibited enzyme activity of the peptidase domain. Interestingly,

32

the activity of LapB was largely increased by various organic solvents such as ethanol,

33

propanol and methanol at the concentration of 60% (v/v). CD analysis provided

34

evidence that organic solvents induce the PA domain conformational changes that

35

eliminate the inhibition role. The unique properties indicate the application potential

36

of LapB in food processing industry.

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38

KEYWORDS

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Aminopeptidase,

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Autoinhibition

organic

solvent-tolerant

enzymes,

Legionella

41 42 43 44 45 46

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pneumophila,

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INTRODUCTION

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Peptidases (EC 3.4) have wide applications in the food, pharmaceutical, detergent,

49

chemical and leather industries.1 Peptidases are a powerful tool in food industry to

50

improve digestibility and solubility, flavor and functional properties of food

51

proteins.2-4 Microbial peptidases represent about 40% of the total enzyme sales

52

worldwide.5 Aminopeptidase (EC 3.4.11) is a peptidase enzyme that catalyzes the

53

removal of N-terminal amino acid residues from peptides and proteins. Bacterial

54

aminopeptidases play a critical role in the catabolism of exogenous peptides, the

55

initial and final steps of protein turnover and the repression of transcription.6

56

Moreover, bacterial aminopeptidases have a high commercial value due to protein

57

hydrolysis for food ingredients and supplements.7 Food protein hydrolysates have a

58

wide range of nutritional applications. In recent years, aminopeptidases from

59

microorganisms have been receiving an increased attention for their applications in

60

food processing. The leucine aminopeptidase rLap1 from Aspergillus sojae improved

61

the degree of hydrolysis of casein and soy protein, and increased the proportions of

62

free amino acids in the hydrolysates and reduce bitter flavour in food processing.3 The

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aminopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 was

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demonstrated in the casein hydrolysis.8 In addition, the leucine aminopeptidase rLAP

65

II from Bacillus stearothermophilus enhance the hydrolysis of anchovy proteins.9

66

Legionella pneumophila, a Gram-negative bacterium, which is in aquatic

67

environments and is able to survive within protozoan cells.10 Type II secretion (T2S)

68

is one of six systems that exist in L. pneumophila for secreting effectors into the

69

extracellular milieu or into host cells.11 Effectors from L. pneumophila T2S can

70

promote bacterial growth under a very wide variety of conditions, ranging from

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extracellular growth at low temperatures to surface translocation, intracellular 3



72

replication in protozoan.12 Owing to the ecological and physiological significance of L.

73

pneumophila T2S, T2S effectors are predicted to function as a potentially important

74

target for industrial and biomedical applications.13 As one of T2S effectors, LapB was

75

identified as extracellular secreted lysine and arginine aminopeptidase from Legionella

76

pneumophila.14 Based on sequence homology search in MEROPS database, LapB has

77

similar characteristics with members from the M28 family of proteases

78

(http://merops.sanger.ac.uk). The M28 family aminopeptidase crystal structures have

79

been solved and reported from Aeromonas proteolytica15, Streptomyces griseus16,

80

human17 and Aneurinibacillus sp. AM-118. According to the previous studies, the M28

81

aminopeptidase family members from different organisms share a common

82

α/β-hydrolase fold with two zinc ions in their active sites. These M28 family

83

aminopeptidases also have a highly conserved nucleophile/histidine/acidic residue

84

triad coordinating zinc ions necessary for proteolytic activity. Sequence alignment

85

reveals that LapB contains a 24-amino acid signal peptide, an N-terminal

86

protease-associated (PA) domain and a C-terminal peptidase domain. Although the

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peptidase domain of LapB is conserved throughout evolution at the amino acid

88

sequence level, biochemical and structural properties of full-length LapB have not

89

been fully understood.

90

Despite a number of commercial aminopeptidases have been used in food industry,

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one of the major challenges in the application of aminopeptidases is discovering its

92

distinctive ability to perform catalysis at extremes of temperature, pH, and organic

93

solvents. Especially for some food fermentation process, ethanol as byproduct will

94

affect the activity of many enzymes. In the present study, we report a crystal structure

95

of extremophilic enzyme LapB from L. pneumophila. Structural and biochemical

96

analyses reveal that the PA domain of LapB shields the active site cavity of the

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C-terminal peptidase domain and inhibit the catalytic activity. More importantly,

98

LapB is found to be an organic solvent-tolerant enzyme. The activity of LapB was

99

largely increased by various organic solvents such as ethanol, propanol and methanol.

100

These properties of LapB indicate the application potential of LapB in nonaqueous

101

biocatalyst and alcoholic fermentation food processing industry.

102

MATERIALS AND METHODS

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Cloning, Expression and Purification

104

DNA for L. pneumophila LapB (NCBI entry code YP_094087) without the

105

24-amino-acid signal peptide was amplified by PCR from L. pneumophila DNA using

106

the following oligonucleotide primers containing artificial BamHI and XhoI sites (in

107

bold):

108

5’-CTACTCGAGCTAATTTAAACCAAGCTCTAC-3’. The PCR product was

109

digested with BamHI and XhoI and was ligated into the BamHI and XhoI sites of the

110

bacterial expression modified pET28a vector (Novagene) with an additional 6×His-

111

glutathione S-transferase (GST) coding sequence following a TEV protease cleavage

112

site at the 5’ end of the gene. DNA for ∆PA-LapB (residues 117-397) (where ∆PA

113

indicates that the N-terminal PA domain is deleted) was amplified by PCR using using

114

the following oligonucleotide primers containing artificial NcoI and XhoI sites (in

115

bold):

116

5’-CCGCTCGAGATTTAAACCAAGCTCTACAATAAAGGCC-3’. The amplified

117

fragments were then cloned into a pET28a vector (Novagen). The cloning junctions

118

were confirmed by DNA sequencing. Each recombinant plasmid was transformed into

119

E. coli strain Rosetta. Cells were grown at 37°C in Luria Bertani (LB) medium

120

containing 100 µg/mL Kanamycin. Expression of LapB and ∆PA-LapB were induced

5’-GAAGGATCCCAGTCCCCTGAAGAAGATAGA-3’and

5’-GAACCATGGCTTACCCGATTAATCATGAAGCACAAG-3’

5


and


121

at an OD600 of 0.8 by adding 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG)

122

followed by incubation at 16°C for 20 h.

123

The cells were harvested and sonicated in 20 mM Tris-HCl buffer pH 8.0 containing

124

200 mM NaCl in an ice-water bath. The lysate was clarified by centrifugation at

125

15000g for 30 min at 5°C. For LapB, the soluble fraction was loaded onto a

126

nickel-chelating resin (Amersham Biosciences) pre-equilibrated with buffer A (20

127

mM Tris-HCl pH 8.0, 200 mM NaCl). The resin was washed with buffer B (20 mM

128

Tris-HCl pH 8.0, 100 mM imidazole, 200 mM NaCl). The fusion protein was eluted

129

with buffer C (20 mM Tris-HCl pH 8.0, 300 mM imidazole, 200 mM NaCl), and was

130

buffer-exchanged with buffer A by centrifugal ultrafiltration (Millipore). The

131

6His-GST-tagged protein was incubated overnight at 5°C with His-tagged TEV

132

protease at a molar ratio of about 20:1. The digested protein was again loaded onto the

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nickel-chelating resin and the flowthrough fraction containing tag-free LapB was

134

collected, concentrated using centrifugal ultrafiltration and subjected to final polishing

135

and buffer exchange by size-exclusion chromatography using a gel-filtration column

136

(Superdex 75 16/60, GE Healthcare) at 15°C in buffer A. For ∆PA-LapB, the

137

supernatant was loaded onto a Ni2+-NTA column (GE Healthcare) equilibrated with

138

buffer A. The column was eluted with buffer C. The sample was subsequently loaded

139

onto a Superdex 75 column (GE Healthcare) equilibrated with buffer A. Protein

140

concentration was determined by the Bradford method (Bio-Rad protein assay) using

141

bovine serum albumin as a standard.19 The presence and purity of the each protein

142

was then analyzed on SDS-PAGE (better than 95% purity).

143

Enzyme Activity Assay

144

The aminopeptidase activities of LapB and ∆PA-LapB were determined by

145

monitoring the hydrolysis L-lysine p-nitroanilide (Lys-pNA) spectrophotometrically

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at 37 °C by monitoring liberated p-nitroanilide.20 The increase in absorbance at 405

147

nm

148

SpectraMaxPlus384). The reaction was performed in buffer (50 mM Tris-HCl, pH

149

8.0), containing 2 mM Lys-pNA and an appropriate amount of enzyme at 37°C for a

150

continuous assay. The enzyme activity unit (U) was defined as the amount of 1 mmol

151

pNA min-1 at 37°C (ε405nm = 8.60 L mmol-1 cm-1). To determine the Km, 10 µL of LapB

152

(42 kDa) (1 mg/mL) or ∆PA-LapB (31 kDa) (0.025 mg/mL) was added to 190 µL of

153

assay buffer (50 mM Tris-HCl, pH 8.0) containing varying concentrations of Lys-pNA

154

(Sigma-Aldrich) (from 0.05 mM to 8 mM) and incubated for 5 min at 37°C. The

155

kinetic parameters were determined by fitting the experimental data to the

156

Michaelis-Menten equation using a nonlinear regression analysis program.

157

To determine the effect of temperature on LapB and ∆PA-LapB activity, enzymatic

158

activity assays were performed in 50 mM Tris-HCl, pH 8.0 at different temperatures

159

ranging from 10 to 90°C at 10°C intervals. To determine the effect of various pH

160

buffers on LapB and ∆PA-LapB activity, assays were performed at 37°C in 50 mM

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citrate-phosphate buffer (pH 3−8), 50 mM potassium phosphate buffer (pH 6−8), 50

162

mM HEPES buffer (pH 6−8), 50 mM Tris-HCl buffer (pH 8−9), and 50 mM

163

Gly-NaOH buffer (pH 9−12). Enzymatic activities in organic solvents were measured

164

by addition of 30% (v/v) or 60% (v/v) organic solvents in 50 mM Tris-HCl, pH 8.0,

165

containing 2-propanol, propanol, ethanol, methanol, acetonitrile. All experiments

166

were performed in triplicates.

167

Crystallization and Data Collection

168

Initial crystallization trials were set up with Crystal Screen, Crystal Screen 2 and

169

PEG/Ion Screen reagent kits (Hampton Research) at 15°C by using the hanging-drop

170

vapour-diffusion method. Each drop, consisting of 1 µL protein solution (5-20 mg/mL)

was

monitored

with

the

spectrophotometry

7


(Molecular

Devices,


171

and an equal volume of reservoir solution, was equilibrated against 200 µL reservoir

172

solution. Further crystal optimization experiments were performed by systematic

173

variation of the precipitant concentration and protein concentration. The LapB crystals

174

were produced by mixing 1µL protein solution (12 mg/mL) and an equal volume of

175

reservoir solution containing 0.1 M sodium citrate tribasic dihydrate pH 6.0, 0.32 M

176

ammonium sulfate, 1.0 M lithium sulfate monohydrate, and incubating at 15°C.

177

The crystals were harvested using cryoloops and immersed briefly in a cryoprotectant

178

solution consisting of 80% (v/v) reservoir solution and 20% (v/v) glycerol. The

179

crystals were subsequently flash-cooled and stored in liquid nitrogen and transferred

180

to beamline BL17U of the SSRF21 (Shanghai Synchrotron Radiation Facility) for

181

X-ray diffraction analysis and data collection.

182

The LapB X-ray data set to 2.5 Å resolution was collected from a single crystal at a

183

wavelength of 0.97931Å. A total of 220 images were recorded with 0.8s exposure

184

using an oscillation range of 1°. The diffraction data were processed with Mosflm and

185

scaled with AIMLESS from the CCP4 program suite.22 The LapB crystal belongs to

186

the space group P42212 with unit-cell parameters a = b = 152.97 Å, c = 108.41 Å.

187

Table 1 gives a summary of the data-collection statistics.

188

Structure Determination and Refinement

189

In order to solve the phase problem, the molecular-replacement method was applied.

190

A. proteolytica aminopeptidase (ApAP, PDB entry 1AMP),15 which has 35% identity

191

to the C-terminal fragment (117-395) of LapB, was used as the search model. The

192

program Phaser23 was used for molecular-replacement calculations. The solution

193

shows two LapB molecules in the crystallographic asymmetric unit. The model was

194

then corrected manually using COOT24 followed by cycles of reciprocal-space

195

refinement with the CCP4 program REFMAC525, which produced an interpretable

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electron density map with the N-terminal domain being especially easily traceable.

197

The CCP4 program Buccaneer26 was then used to extend the initial model, which

198

allowed automatic building of most residues of N-terminal domain. Additional

199

missing residues in the auto-built model were added in COOT manually.24 The

200

structure was refined with REFMAC25 and PHENIX27.

201

The final model contains 732 residues, 46 water molecules, 2 sulfate ions and four

202

zinc ions in the asymmetric unit. The three N-terminal residues and two regions

203

(residues 111-114 on chain A and chain B) were not modeled because of the poor

204

electron density. The quality of model was evaluated using MolProbity2 with all

205

parameters within the expected value range at the resolutions. The refinement

206

statistics are summarized in Table 1. The coordinates and structure factors have been

207

deposited in the Protein Data Bank under the accession code 5GNE.

208

Circular Dichroism (CD) Spectroscopic Analysis

209

The CD spectra of LapB and ∆PA-LapB were recorded at 25°C with a

210

spectropolarimeter (BioLogic, MOS-500) using a quartz cuvette with a path length of

211

0.1 cm. The protein sample was dissolved to 0.1 mg/mL in 50 mM potassium

212

phosphate buffer pH 8.0. To estimate the secondary structure change of LapB and

213

∆PA-LapB, the protein samples were measured at 25°C after incubating it with 20%

214

(v/v) or 60% (v/v) organic solvent (2-propanol, propanol, ethanol, methanol). All

215

experiments were performed in triplicates. Secondary structures were estimated using

216

the Dichroprot 2000 1.0.4 software.

217

RESULTS AND DISCUSSION

218

Overall Structure of LapB

219

The crystal of LapB diffracts to 2.5 Å resolution and belongs to the P42212 space

220

group, with two molecules in an asymmetric unit. Analysis using PISA28 suggested 9



221

that monomer is likely to be the biologically relevant form of the protein. This was

222

consistent with LapB being monomeric state in solution using size-exclusion

223

chromatography (Supporting Information Figure S1). The LapB consists of an

224

N-terminal protease-associated (PA) domain (amino acids 25-106) and a C-terminal

225

peptidase domain (amino acids 117-397) connected by a long flexible loop (residues

226

107-116) (Figure 1A). The structure of the two molecules in the asymmetric unit is

227

essentially similar, with RMSD (root mean square deviations) of 0.287 Å.

228

The peptidase domain, similar to A. proteolytica aminopeptidase (ApAP),15 folds into

229

a single mixed α/β globular structure with a twisted central eight-stranded mixed

230

parallel and anti-parallel β-sheet core sandwiched between three and six helices. The

231

peptidase domain is responsible for catalysis and binds the two zinc ions. The pair of

232

metal ions is located between the PA domain and the peptidase domain, close to the

233

edge of the β-sheet core. Similar to other aminopeptidases, the pair of zinc ions is

234

coordinated by a water molecule and the side chains of several conserved residues,

235

His213, Asp226, Glu260, Glu261, Asp288 and His366 (Figure 1B). In M28 family,

236

non-catalytic domain designated as PA domain was found in some aminopeptidases.

237

The PA domain found in other peptidases is thought to assist the proper folding of

238

enzymes, function as an inhibitor of the cognate mature enzymes, mediate protein

239

sorting and interact with other molecules or proteins.29 Interestingly, Arg74 from the

240

PA domain also makes water-mediated hydrogen bonds to the dinuclear zinc ions by

241

the water molecule. The active site cavity of the C-terminal peptidase domain is partly

242

shielded by cap-like PA domain, suggesting that the PA domain may affect LapB

243

peptidase activity.

244

Active Site of LapB

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The peptidase domain of LapB has 35% and 31% sequence identity to ApAP from A.

246

proteolytica and rLap1 from A. sojae, respectively. Structure-based alignment of

247

LapB and ApAP revealed that the six conserved amino acid residues (His213,

248

Asp226, Glu260, Glu261, Asp288 and His366) from LapB involved in the

249

coordination of the two zinc ions and one water molecule are critical for catalysis

250

(Figure 2A). His213, His366 and Glu261 form a catalytic triad. The conserved

251

residue Glu260 makes water-mediated hydrogen bonds to the zinc ions. According to

252

the proposed reaction mechanism of ApAP,30 the conserved residue Glu260 in LapB

253

may function as the general base since it forms a hydrogen bond to the bridging

254

water. The zinc-bound water molecule is probably displaced by substrate, or

255

functions as the attacking nucleophile at the carbonyl carbon of the peptide bound.

256

Asp226 and Asp288 are also important for stabilization of His213 coordinating the

257

zinc ion via hydrogen bonds. His366 may stabilize the transition state of substrate.

258

In the structure of ApAP complexed with bestatin (PDB code 1XRY),30 Asp117,

259

Glu151, Glu152, Asp179, Tyr225, Cys227, Phe244 and Tyr251 are essential for

260

substrate binding and stabilize the transition state. The analogous positions for these

261

residues in LapB are occupied by Asp226, Glu260, Glu261, Asp288, Tyr335, Cys337,

262

Ser354, Asp361 (Figure 2B). Structural comparison of LapB and ApAP substrate

263

binding sites showed that the orientation and conformation of Tyr335, Ser354 and

264

Asp361 in LapB are totally different from the corresponding Tyr225, Phe244 and

265

Tyr251 in ApAP. Compared to the conserved residue Tyr225 in ApAP, the

266

corresponding Tyr335 in LapB apparently undergoes near 180° rotation, and is

267

protruding out of the active site. In ApAP-bestatin complex structure,30 the pyridine

268

ring of bestatin forms π-π stacking between Phe244 and Tyr251. The relevant residues

269

in LapB are substituted by Ser354 and Asp361. These different side chains are

11



270

composed of a larger substrates binding pocket with more negative charge than that in

271

the ApAP structure (Figure 2C). It may illustrate the substrate specificity of LapB with

272

preference for positively charged, hydrophilic substrates14.

273

The PA Domain Plays a Crucial Role in an Autoinhibitory Mechanism of LapB

274

The PA domain of LapB also has a single mixed α/β structure, but with a

275

four-stranded antiparallel β-sheet sandwiched between several helices of different

276

length. α2, connecting with α1 by disulfide bridge (Cys37-Cys77), occupies a position

277

close to the active site (Figure 3A). A structural homology search using DALI

278

revealed that PA domain has a novel fold, different from those of other known folds in

279

the database. The interactions between the PA domain and C-terminal peptidase

280

domain of LapB are contributed by hydrogen bonds involving residues Arg74, Asn79,

281

Tyr80, Asn82, Glu84, Tyr86, Leu104 from the N-terminal PA domain, and Arg262,

282

Gly263, Tyr269, Tyr335, Ala336, Cys337, Asn342, Asn345 from the C-terminal

283

peptidase domain (Figure 3B). In addition, the orientation of α2 from the PA domain

284

allows Arg74 to point into the active site region. Arg74 with a long positive charged

285

side chain contacts zinc ion via the zinc-bound water molecule, which facilitates the

286

PA domain covering the active site pocket. The interactions between Arg74 and the

287

zinc-bound water may mimic the transition state of substrate binding. Structural

288

analysis of LapB suggested that the PA domain sterically shields the entrance of active

289

site from C-terminal peptidase domain and prevents direct access to the substrate

290

binding site. It implied that the PA domain may be responsible for regulating

291

enzymatic activity as an auto-inhibitor.

292

To investigate the possible role of the PA domain, ∆PA-LapB was expressed and

293

purified for enzymatic activity examination. The specific activity toward Lys-pNA of

294

LapB was 0.360±0.018 U/mg, which was much smaller than that of ∆PA-LapB

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(16.80±0.24 U/mg). The observed Km of LapB and ∆PA-LapB for Lys-pNA were

296

determined to be 0.732±0.130 mM and 0.195±0.023 mM, respectively (Table 2).

297

These results suggest that the PA domain inhibits substrate binding of the C-terminal

298

peptidase domain. In addition, the observed Kcat/Km value of ∆PA-LapB increase

299

compared to that of LapB, conforming that the PA domain inhibits the activity of the

300

C-terminal peptidase domain. It’s most likely that the PA domain has not completely

301

prevented the entrance of the substrate to the active site, resulting in the activity of

302

full-length LapB observed. The finding that the PA domain inhibits hydrolytic activity

303

of LapB in the catalytic process is consistent with the structural orientation of the PA

304

domain. It means that the activity site of LapB may be “closed” state or “open” state

305

when with or without the PA domain. The kinetic and structural analysis implied that

306

the PA domain contributes to reduce catalytic efficiency by partly covering the active

307

site pocket.

308

Effect of Temperature, pH and Organic Solvent on the Enzymatic Activity of

309

LapB

310

To investigate the effect of temperature, the enzyme activities of LapB and ∆PA-LapB

311

were tested at various temperatures. LapB and ∆PA-LapB exhibited maximum activity

312

at around 60°C (relative activity set to 100%). The ∆PA-LapB was almost inactivated

313

when the temperature reached 80°C, while LapB still retained 10% of its maximal

314

activity at 90°C (Figure 4A). The enzyme activities of LapB and ∆PA-LapB were

315

determined at different pH values (Figure 4B). ∆PA-LapB showed the maximum

316

hydrolytic activity at pH 8.0 (relative activity set to 100%). However, LapB showed

317

highest activity at pH 11 (relative activity set to 100%). It indicates that LapB is more

318

resistant to the effects of extreme alkaline pH than ∆PA-LapB. These results revealed

319

that the PA domain contributes to the stabilization of LapB. The attachment of the

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320

cap-like PA domain to the peptidase domain may be one of the strategies of LapB to

321

adapt to high-temperature and extreme environment.

322

The majority of functional proteins are known to denature and lose their biological

323

activity in organic solvent. Interestingly, both LapB and ∆PA-LapB show high

324

resistance to the effect of organic solvents. The activity of LapB and ∆PA-LapB were

325

measured at 37°C in reaction mixtures containing various concentrations of different

326

organic solvents, like acetonitrile, ethanol, methanol, propanol and 2-propanol (Figure

327

4C). In the buffer without organic solvents, LapB exhibited a relatively low activity

328

due to the autoinhibitory property of the PA domain, and deleting the PA domain of

329

LapB resulted in an increase in its basal activity. The relative activity of LapB and

330

∆PA-LapB were taken as 100% in the buffer without organic solvents, respectively. In

331

the presence of 20% (v/v) acetonitrile, ethanol, methanol, propanol and 2-propanol,

332

LapB retained more than 50% residual activity. Moreover, the activity of LapB

333

increased approximately six fold in 60% (v/v) ethanol, 2-propanol and propanol. It

334

suggested that the activation of LapB activity can be induced by organic solvents.

335

Compared with LapB, ∆PA-LapB has relatively low tolerance to organic solvents.

336

∆PA-LapB exhibited a decrease in activity at an increase in methanol, propanol,

337

2-propanol and acetonitrile concentration. However, ∆PA-LapB showed an increased

338

activity even in 20% (v/v) of ethanol. Meanwhile, these results show that LapB and

339

∆PA-LapB all display high resistance to organic solvents especially like alcohols. A

340

unique characteristic of LapB is the increase of enzyme activity in presence of organic

341

solvents. Organic solvent-tolerant enzymes are expected to have potential applications

342

in industrial chemical processes. To date, there were some reports that organic

343

solvent-tolerant peptidases from Pseudomonas aeruginosa strains are stable in the

344

presence of 20% (v/v) ethanol, methanol and 2-propanol.31,

14


32

A peptidase from

Page 15 of 35


345

Fasciola hepatica was stable in the presence of 30% (v/v) methanol.33 The organic

346

solvent-activated LapB and organic solvent-tolerant ∆PA-LapB retained more than

347

60% of their initial activity after exposure to 60% (v/v) ethanol, which could

348

potentially be used as an enzyme additive in fermented foods.

349

The Organic Solvents Effect on the Secondary Structure of LapB

350

The analysis of CD spectra reflected changes in secondary structure of LapB and

351

∆PA-LapB in the presence of various organic solvents (Figure 5). As the

352

concentration of ethanol, methanol, 2-propanol and propanol increased to 60% (v/v),

353

the CD spectrum of LapB showed the shape of the mainly α-helical secondary

354

structure of the negative ellipticity bands near 222 nm and 208 nm and a positive band

355

at 194 nm. Moreover, the proportion of α-helix increased largely and that of random

356

coil and β-sheet decreased greatly with increasing concentrations of these organic

357

solvents. Ethanol, methanol, 2-propanol and propanol can induce the formation of

358

secondary structures (especially α-helix) of LapB, and a decrease of β-sheet content.

359

Compared to the secondary structure of LapB, ∆PA-LapB showed only a slight

360

increase in α-helix content with increased concentration of ethanol, methanol,

361

2-propanol and propanol, respectively (Figure 6). However, the β-sheet of ∆PA-LapB

362

remained unchanged in the presence of those organic solvents, which means that these

363

organic solvents mainly cause the change of the β-sheet content of the PA domain. We

364

proposed that a hydrophobic phase may induce a conformational change of the PA

365

domain, and will induce opening of the PA domain to make the active site of LapB

366

more accessible. Organic solvent-induced secondary structural changes are correlated

367

to the enzymatic activity. These studies suggest that LapB undergoes a large

368

conformational change and gains a catalytically active state in the presence of organic

369

solvents, and indicate that the structure and activity transition of LapB may be

15



370

modulated by the organic solvent. Due to its unique characteristic, LapB could be

371

therefore applied in nonaqueous biocatalyst and food processing industry.

372

ACKNOWLEDGEMENTS

373

The authors thank Professor Zhaoqing Luo (Purdue University) for L. pneumophila

374

genomic DNA. We also thank the staff at SSRF beamline BL17U for assistance with

375

synchrotron data collection. This work was supported by grants from the National

376

Natural Science Foundation of China (31400641, 31270770), Anhui International

377

Cooperation Project (1704e1002228) and the Science Foundation of Anhui University

378

(J18520219, KYXL2016105).

379

SUPPORTING INFORMATION

380

Figure S1. Analytical gel filtration chromatography

381

16


Page 16 of 35

Page 17 of 35


382

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383

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rLap1 from Aspergillus sojae and Its Application in Debittering. Appl Biochem

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Biotechnol 2015, 177, 190-206.

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Singh, R.; Kumar, M.; Mittal, A.; Mehta, P. K., Microbial enzymes: industrial

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Stressler, T.; Eisele, T.; Schlayer, M.; Lutz-Wahl, S.; Fischer, L., Characterization

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characterisation, and structure analysis of Bacillus subtilis aminopeptidase. J Sci Food

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10. Lau, H. Y.; Ashbolt, N. J., The role of biofilms and protozoa in Legionella

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11. DebRoy, S.; Dao, J.; Soderberg, M.; Rossier, O.; Cianciotto, N. P., Legionella

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12. Cianciotto, N. P., Type II secretion: a protein secretion system for all seasons.

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13. Cianciotto, N. P., Many substrates and functions of type II secretion: lessons

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learned from Legionella pneumophila. Future Microbiol 2009, 4, 797-805.

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14. Rossier, O.; Dao, J.; Cianciotto, N. P., The type II secretion system of Legionella

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pneumophila elaborates two aminopeptidases, as well as a metalloprotease that

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15. Chevrier, B.; Schalk, C.; D'Orchymont, H.; Rondeau, J. M.; Moras, D.; Tarnus,

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C., Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical

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member of the co-catalytic zinc enzyme family. Structure 1994, 2, 283-91.

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16. Gilboa, R.; Greenblatt, H. M.; Perach, M.; Spungin-Bialik, A.; Lessel, U.;

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Wohlfahrt, G.; Schomburg, D.; Blumberg, S.; Shoham, G., Interactions of

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Streptomyces griseus aminopeptidase with a methionine product analogue: a

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structural study at 1.53 A resolution. Acta Crystallogr D Biol Crystallogr 2000, 56,

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17. Davis, M. I.; Bennett, M. J.; Thomas, L. M.; Bjorkman, P. J., Crystal structure of

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18. Akioka, M.; Nakano, H.; Horikiri, A.; Tsujimoto, Y.; Matsui, H.; Shimizu, T.;

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Nakatsu, T.; Kato, H.; Watanabe, K., Overexpression, purification, crystallization and

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preliminary X-ray crystallographic studies of a proline-specific aminopeptidase from

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Aneurinibacillus sp. strain AM-1. Acta Crystallogr Sect F Struct Biol Cryst Commun

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2006, 62, 1266-8.

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19. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram

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quantities of protein utilizing the principle of protein-dye binding. Anal Biochem

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1976, 72, 248-54.

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20. Tuppy, H.; Wiesbauer, U.; Wintersberger, E., [Amino acid-p-nitroanilide as a

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substrate for aminopeptidases and other proteolytic enzymes]. Hoppe Seylers Z

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Physiol Chem 1962, 329, 278-88.

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21. Wang, Z.; Pan, Q.; Yang, L.; Zhou, H.; Xu, C.; Yu, F.; Wang, Q.; Huang, S.; He,

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J., Automatic crystal centring procedure at the SSRF macromolecular crystallography

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beamline. J Synchrotron Radiat 2016, 23, 1323-1332.

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22. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J., Refinement of macromolecular

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structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr

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1997, 53, 240-55.

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23. McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L.

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C.; Read, R. J., Phaser crystallographic software. J Appl Crystallogr 2007, 40,

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658-674.

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24. Emsley, P.; Cowtan, K., Coot: model-building tools for molecular graphics. Acta

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Crystallogr D Biol Crystallogr 2004, 60, 2126-32.

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25. Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.;

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Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A., REFMAC5 for the refinement of

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macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 2011, 67,

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355-67.

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26. Cowtan, K., The Buccaneer software for automated model building. 1. Tracing

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protein chains. Acta Crystallogr D Biol Crystallogr 2006, 62, 1002-11.

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27. Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.;

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Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.;

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Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.;

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Terwilliger, T. C.; Zwart, P. H., PHENIX: a comprehensive Python-based system for

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macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 2010, 66,

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213-21.

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28. Krissinel, E.; Henrick, K., Inference of macromolecular assemblies from

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crystalline state. J Mol Biol 2007, 372, 774-97.

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29. Demidyuk, I. V.; Shubin, A. V.; Gasanov, E. V.; Kostrov, S. V., Propeptides as

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modulators of functional activity of proteases. Biomol Concepts 2010, 1, 305-22.

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30. Chevrier, B.; D'Orchymont, H.; Schalk, C.; Tarnus, C.; Moras, D., The structure

473

of the Aeromonas proteolytica aminopeptidase complexed with a hydroxamate

474

inhibitor. Involvement in catalysis of Glu151 and two zinc ions of the co-catalytic

475

unit. Eur J Biochem 1996, 237, 393-8.

476

31. Ogino, H.; Yokoo, J.; Watanabe, F.; Ishikawa, H., Cloning and sequencing of a

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gene of organic solvent-stable protease secreted from Pseudomonas aeruginosa

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PST-01 and its expression in Escherichia coli. Biochem Eng J 2000, 5, 191-200.

479

32. Gupta, A.; Roy, I.; Khare, S. K.; Gupta, M. N., Purification and characterization

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of a solvent stable protease from Pseudomonas aeruginosa PseA. J Chromatogr A

481

2005, 1069, 155-61.

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33. Dowd, A. J.; Dooley, M.; Fagain, C.; Dalton, J. P., Stability studies on the

483

cathepsin L proteinase of the helminth parasite, Fasciola hepatica. Enzyme Microb

484

Technol 2000, 27, 599-604.

485

34. Corpet, F., Multiple sequence alignment with hierarchical clustering. Nucleic

486

Acids Res 1988, 16, 10881-90.

21



487

35. Gouet, P.; Robert, X.; Courcelle, E., ESPript/ENDscript: Extracting and rendering

488

sequence and 3D information from atomic structures of proteins. Nucleic Acids Res

489

2003, 31, 3320-3.

490

22


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491

Figure 1. (A) Cartoon representation of LapB structure. The N-terminal PA domain

492

and the C-terminal peptidase domain are in magenta and green, respectively. The

493

disordered region (residues 111-114) is shown as a dashed line. Zinc ions are drawn as

494

spheres in grey. (B) Multiple alignment of LapB, ApAP (PDB code 1AMP) and S.

495

griseus aminopeptidase (PDB code 1CP7). The alignment is performed using

496

MultAlin34 and ESPript35. The secondary structural elements of LapB are displayed at

497

the top of the alignment. The α-helices, 310-helices, β-strands and strict β-turns are

498

denoted as α, η, β and TT, correspondingly. The residues of the signal peptide, the

499

N-terminal PA domain and the predicted substrate-binding site of LapB are marked in

500

light yellow, magenta and green respectively. The substrate-binding residues are

501

markered in light green. The conserved active site residues are marked by a filled blue

502

triangle.

503

Figure 2. (A) Superposition of the active site of LapB (green) and ApAP (PDB code

504

1AMP) (orange). The active site residues are shown in ball-stick. The zinc ions and

505

water moleculer from LapB are shown as spheres and colored in grey and violet,

506

respectively. The zinc ions from ApAP (PDB code 1AMP) are shown as slate spheres.

507

(B) Comparison of LapB (green) and ApAP complex with bestatin (PDB code 1XRY)

508

(yellow) structures in the region of substrate-binding site. The bestatin binding

509

residues from ApAP complex with bestatin (PDB code 1XRY) are shown as ball-stick

510

and colored in yellow. The corresponding residues of LapB, which are thought to be

511

involved in catalysis, are shown as ball-stick and colored in green. The zinc ions and

512

water molecule from LapB are shown as spheres and colored in grey and violet,

513

respectively. The zinc ions from ApAP complex with bestatin (PDB code 1XRY) are

514

shown as slate spheres. (C) The electrostatic surface plot of the C-terminal peptidase 23



515

domain of LapB (left) and ApAP (PDB code 1AMP) (right) show the substrate

516

binding pocket (black circle). The surface is colored based on electrostatic potential

517

with positively charged regions in blue and the negatively charged regions in red.

518

Figure 3. The interaction between the N-terminal PA domain and the C-terminal

519

peptidase domain of LapB. (A) Stereo view shows that the PA domain partly shields

520

the active pocket. The N-terminal PA domain and the C-terminal peptidase domain are

521

shown as cartoon and surface, respectively. The zinc ions and water moleculer from

522

LapB are shown as spheres and colored in grey and violet, respectively. (B) Stereo

523

view shows that the residues from N-terminal PA domain (colored in magenta) and

524

C-terminal peptidase domain (colored in green) involved in the interactions are shown

525

in stick form. The specific interactions block residue Arg74 coordinating with the

526

active site water involved in the substrate recognition. The coordinate bonds are

527

indicated as magenta dotted lines.

528

Figure 4. Effects of pH, temperature and organic solvent on LapB and ∆PA-LapB. (A)

529

Effect of temperature on LapB and ∆PA-LapB. Relative activity is shown as that

530

against the highest activity (100%). (B) Effect of pH on LapB and ∆PA-LapB.

531

Relative activity is shown as that against the highest activity (100%). (C) Effect of

532

organic solvent on LapB and ∆PA-LapB. The activity of the control (without organic

533

solvent) was taken as 100 %.

534

Figure 5. Far UV CD spectra of LapB (left) and ∆PA-LapB (right) in the presence of

535

20% and 60% organic solvents.

536

Figure 6. The secondary structures of LapB (left) and ∆PA-LapB (right) in the

537

presence of 20% and 60% organic solvents.

538 539

24


Page 24 of 35

Page 25 of 35


Table 1. Data Collection and Refinement Statistics. LapB SSRF beamline

BL17U

Wavelength (Å)

0.97931

Space group

P42212

Molecules/ASU

2

Cell parameters a/b/c (Å)

152.97/152.97/108.41

α/β/γ (°)

90.00/90.00/90.00

Resolution range (Å)

48-2.5(2.59-2.5)

No. of unique reflections

42854 (4641)

Rp.i.m.a (%)

4.0(42.5)

Average I/σ(I)

18.9 (3.0)

CC1/2

99.9 (83.7)

Redundancy

17.4(17.8)

Completeness (%)

95.1 (100)

Refinement statistics R-factorb (%)

18.0

R-freec (%)

22.7

RMSDd bond length (Å)

0.010

25



RMSD bond angles (°)

1.41

Number of protein

5843

atoms/ASU 46

Number of water atoms/ASU

Ramachandran plote (%) Ramachandran favored

96

Ramachandran Outliers

0

PDB code

5GNE

Values in parentheses are for the highest resolution shell. a

Rp.i.m = ∑hkl [1/(nhkl – 1)]1/2∑i |Ii(hkl) – 〈I(hkl)〉|/ ∑hkl ∑iIi(hkl), where nhkl is the

number of observations of reflection hkl. b

R-factor =Σh | |Fobs|－|Fcalc| |/Σ|Fobs|, where |Fobs| and |Fcalc| are the observed and

calculated structure factor amplitudes, respectively. Summation includes all reflections used in the refinement. c

Free R factor = Σ| |Fobs|－|Fcalc| |/Σ|Fobs|, evaluated for a randomly chosen subset of

5% of the diffraction data not included in the refinement. d

e

Root-mean square-deviation from ideal values.

Categories were defined by Molprobity.

26


Page 26 of 35

Page 27 of 35


Table 2. Kinetic Parameters of LapB and ∆PA-LapB for Substrate Lys-pNA. Enzyme

Km

Kcat

Kcat/Km

Vmax

(mM)

(s-1)

(mM−1 s−1)

(mM min−1 mg−1)

LapB

0.732±0.130

0.253±0.013

0.345±0.099

1.8+0. 091

∆PA-LapB

0.195±0.023

9.008±0.148

46±6.296

84+1.231

27



1

28


Page 28 of 35

Page 29 of 35


Figure 1. (A) Cartoon representation of LapB structure. The N-terminal PA domain and the C-terminal peptidase domain are in magenta and green, respectively. The disordered region (residues 111-114) is shown as a dashed line. Zinc ions are drawn as spheres in grey. (B) Multiple alignment of LapB, ApAP (PDB code 1AMP) and S. griseus aminopeptidase (PDB code 1CP7). The alignment is performed using MultAlin34 and ESPript35. The secondary structural elements of LapB are displayed at the top of the alignment. The αhelices, 310-helices, β-strands and strict β-turns are denoted as α, η, β and TT, correspondingly. The residues of the signal peptide, the N-terminal PA domain and the predicted substrate-binding site of LapB are marked in light yellow, magenta and green respectively. The substrate-binding residues are markered in light green. The conserved active site residues are marked by a filled blue triangle. 129x168mm (300 x 300 DPI)



Page 30 of 35

Figure 2. (A) Superposition of the active site of LapB (green) and ApAP (PDB code 1AMP) (orange). The active site residues are shown in ball-stick. The zinc ions and water moleculer from LapB are shown as spheres and colored in grey and violet, respectively. The zinc ions from ApAP (PDB code 1AMP) are shown as slate spheres. (B) Comparison of LapB (green) and ApAP complex with bestatin (PDB code 1XRY) (yellow) structures in the region of substrate-binding site. The bestatin binding residues from ApAP complex with bestatin (PDB code 1XRY) are shown as ball-stick and colored in yellow. The corresponding residues of LapB, which are thought to be involved in catalysis, are shown as ball-stick and colored in green. The zinc ions and water molecule from LapB are shown as spheres and colored in grey and violet, respectively. The zinc ions from ApAP complex with bestatin (PDB code 1XRY) are shown as slate spheres. (C) The electrostatic surface plot of the C-terminal peptidase domain of LapB (left) and ApAP (PDB code 1AMP) (right) show the substrate binding pocket (black circle). The surface is colored based on electrostatic potential with positively charged regions in blue and the negatively charged regions in red. 101x103mm (300 x 300 DPI)


Page 31 of 35


Figure 3. The interaction between the N-terminal PA domain and the C-terminal peptidase domain of LapB. (A) Stereo view shows that the PA domain partly shields the active pocket. The N-terminal PA domain and the C-terminal peptidase domain are shown as cartoon and surface, respectively. The zinc ions and water moleculer from LapB are shown as spheres and colored in grey and violet, respectively. (B) Stereo view shows that the residues from N-terminal PA domain (colored in magenta) and C-terminal peptidase domain (colored in green) involved in the interactions are shown in stick form. The specific interactions block residue Arg74 coordinating with the active site water involved in the substrate recognition. The coordinate bonds are indicated as magenta dotted lines. 109x120mm (300 x 300 DPI)



Figure 4. Effects of pH, temperature and organic solvent on LapB and ∆PA-LapB. (A) Effect of temperature on LapB and ∆PA-LapB. Relative activity is shown as that against the highest activity (100%). (B) Effect of pH on LapB and ∆PA-LapB. Relative activity is shown as that against the highest activity (100%). (C) Effect of organic solvent on LapB and ∆PA-LapB. The activity of the control (without organic solvent) was taken as 100 %. 110x136mm (300 x 300 DPI)


Page 32 of 35

Page 33 of 35


Figure 5. Far UV CD spectra of LapB (left) and ∆PA-LapB (right) in the presence of 20% and 60% organic solvents. 130x196mm (300 x 300 DPI)



Figure 6. The secondary structures of LapB (left) and ∆PA-LapB (right) in the presence of 20% and 60% organic solvents. 126x193mm (300 x 300 DPI)


Page 34 of 35

Page 35 of 35


TOC Graphic 70x45mm (300 x 300 DPI)


Crystal structure and biochemical characterization of an

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