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Article
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
2
aminopeptidase LapB from Legionella pneumophila
3
Nannan Zhang, Shiyan Yin, Wei Zhang, Xiaojian Gong, Na Zhang, Kai Fang,
4
Honghua Ge *
5 6
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] 13
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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
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of LapB in food processing industry.
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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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INTRODUCTION
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Peptidases (EC 3.4) have wide applications in the food, pharmaceutical, detergent,
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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
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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
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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
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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
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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)
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is one of six systems that exist in L. pneumophila for secreting effectors into the
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extracellular milieu or into host cells.11 Effectors from L. pneumophila T2S can
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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
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replication in protozoan.12 Owing to the ecological and physiological significance of L.
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pneumophila T2S, T2S effectors are predicted to function as a potentially important
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target for industrial and biomedical applications.13 As one of T2S effectors, LapB was
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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
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aminopeptidase family members from different organisms share a common
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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
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sequence level, biochemical and structural properties of full-length LapB have not
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been fully understood.
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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
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distinctive ability to perform catalysis at extremes of temperature, pH, and organic
93
solvents. Especially for some food fermentation process, ethanol as byproduct will
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affect the activity of many enzymes. In the present study, we report a crystal structure
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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,
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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.
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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
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E. coli strain Rosetta. Cells were grown at 37°C in Luria Bertani (LB) medium
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containing 100 µg/mL Kanamycin. Expression of LapB and ∆PA-LapB were induced
5’-GAAGGATCCCAGTCCCCTGAAGAAGATAGA-3’and
5’-GAACCATGGCTTACCCGATTAATCATGAAGCACAAG-3’
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at an OD600 of 0.8 by adding 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG)
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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
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nickel-chelating resin (Amersham Biosciences) pre-equilibrated with buffer A (20
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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
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with buffer C (20 mM Tris-HCl pH 8.0, 300 mM imidazole, 200 mM NaCl), and was
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buffer-exchanged with buffer A by centrifugal ultrafiltration (Millipore). The
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6His-GST-tagged protein was incubated overnight at 5°C with His-tagged TEV
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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
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collected, concentrated using centrifugal ultrafiltration and subjected to final polishing
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and buffer exchange by size-exclusion chromatography using a gel-filtration column
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(Superdex 75 16/60, GE Healthcare) at 15°C in buffer A. For ∆PA-LapB, the
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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).
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Enzyme Activity Assay
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The aminopeptidase activities of LapB and ∆PA-LapB were determined by
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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
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nm
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SpectraMaxPlus384). The reaction was performed in buffer (50 mM Tris-HCl, pH
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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
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(42 kDa) (1 mg/mL) or ∆PA-LapB (31 kDa) (0.025 mg/mL) was added to 190 µL of
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assay buffer (50 mM Tris-HCl, pH 8.0) containing varying concentrations of Lys-pNA
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(Sigma-Aldrich) (from 0.05 mM to 8 mM) and incubated for 5 min at 37°C. The
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kinetic parameters were determined by fitting the experimental data to the
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Michaelis-Menten equation using a nonlinear regression analysis program.
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To determine the effect of temperature on LapB and ∆PA-LapB activity, enzymatic
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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
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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.
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Crystallization and Data Collection
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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
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and an equal volume of reservoir solution, was equilibrated against 200 µL reservoir
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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.
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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
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to beamline BL17U of the SSRF21 (Shanghai Synchrotron Radiation Facility) for
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X-ray diffraction analysis and data collection.
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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
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the space group P42212 with unit-cell parameters a = b = 152.97 Å, c = 108.41 Å.
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Table 1 gives a summary of the data-collection statistics.
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Structure Determination and Refinement
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In order to solve the phase problem, the molecular-replacement method was applied.
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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
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program Phaser23 was used for molecular-replacement calculations. The solution
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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.
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The CCP4 program Buccaneer26 was then used to extend the initial model, which
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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
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structure was refined with REFMAC25 and PHENIX27.
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The final model contains 732 residues, 46 water molecules, 2 sulfate ions and four
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zinc ions in the asymmetric unit. The three N-terminal residues and two regions
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(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.
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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.
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RESULTS AND DISCUSSION
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Overall Structure of LapB
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The crystal of LapB diffracts to 2.5 Å resolution and belongs to the P42212 space
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group, with two molecules in an asymmetric unit. Analysis using PISA28 suggested 9
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that monomer is likely to be the biologically relevant form of the protein. This was
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consistent with LapB being monomeric state in solution using size-exclusion
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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.
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proteolytica and rLap1 from A. sojae, respectively. Structure-based alignment of
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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.
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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
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composed of a larger substrates binding pocket with more negative charge than that in
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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
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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
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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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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.
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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,
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A peptidase from
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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
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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
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Stressler, T.; Eisele, T.; Schlayer, M.; Lutz-Wahl, S.; Fischer, L., Characterization
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22. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J., Refinement of macromolecular
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26. Cowtan, K., The Buccaneer software for automated model building. 1. Tracing
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Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.;
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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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30. Chevrier, B.; D'Orchymont, H.; Schalk, C.; Tarnus, C.; Moras, D., The structure
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of the Aeromonas proteolytica aminopeptidase complexed with a hydroxamate
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inhibitor. Involvement in catalysis of Glu151 and two zinc ions of the co-catalytic
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unit. Eur J Biochem 1996, 237, 393-8.
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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.
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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
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33. Dowd, A. J.; Dooley, M.; Fagain, C.; Dalton, J. P., Stability studies on the
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cathepsin L proteinase of the helminth parasite, Fasciola hepatica. Enzyme Microb
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Technol 2000, 27, 599-604.
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34. Corpet, F., Multiple sequence alignment with hierarchical clustering. Nucleic
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Acids Res 1988, 16, 10881-90.
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35. Gouet, P.; Robert, X.; Courcelle, E., ESPript/ENDscript: Extracting and rendering
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sequence and 3D information from atomic structures of proteins. Nucleic Acids Res
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2003, 31, 3320-3.
490
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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
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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
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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
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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.
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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
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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)
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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)
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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)
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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)
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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)
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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)
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TOC Graphic 70x45mm (300 x 300 DPI)
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