Chapter 1. Introduction
1.9 Thesis purpose
Aminoacylhistidine dipeptidase is widely distributed among prokaryotes and eukaryotes, and its biological significance and function have been evolutionarily conserved as well. PepD is well known to hydrolyze physiologically-important dipeptides into two amino acids, such as carnosine and homocarnosine, but the structure-reaction relationship of PepD has yet to be studied in detail. V. alginolyticus PepD shares only 20% amino acid sequence identity with the several other M20 family enzymes whose crystal structures have been reported76. In our previous studies, we had cloned and expressed the V. alginolyticus pepD gene and were able to carry out detailed
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biochemical characterization, including defining the enzyme kinetics of the produced recombinant protein76. The subsequent studies that comprised this thesis aimed at investigating the structure-function relationship by using protein X-ray crystallography coupled with site-directed mutagenesis77, 78. Enzyme kinetic studies were also carried out to determine the reaction rate and underlying catalytic mechanism. Ultimately, our characterization of the active-site architecture of PepD enzyme will aid in future studies to identify residues that may be modified to yield alternative substrate recognition properties and improve the potential therapeutic value of this protein and its closely related family members.
On the other hand, previous studies have reported that substituting zinc ions for copper ions in the active site of aminopeptidases allowed for the enzyme function to be effectively converted from peptide hydrolase to catechol oxidase72. Therefore, the copper-substituted PepD (CuCu-PepD) was prepared to investigate whether conversion of enzyme function could be attained. Moreover, the robustness of the new activity was evaluated, as well as the new substrate selectivity, by using several catechol derivatives.
The findings from this study are expected to provide further insights into the general enzyme activities of peptide hydrolysis and catechol oxidation, and advance our understanding of the correlation between specific metal ions and enzyme function.
Finally, this study might also reveal a new direction for divergent enzyme evolution.
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CHAPTER 2
Materials and Methods
2.1 Materials
2.1-1 Bacterial strains, molecular cloning/expression vectors, and resin
In this study, bacterial strains Escherichia coli XL1-Blue, BL21(DE3) and BL21(DE3) pLysS were purchased from Novagen. Plasmid vector pET-28a(+) and pCR®2.1-TOPO were purchased from Invitrogen and Novagen, respectively. Bacterial growth condition, agar plate preparation, recombinant DNA purification, DNA sequence determination, agarose gel electrophoresis and protein concentration determination were performed according to standard procedures or the commercial kit. Ni-NTA His-Band® Resin was purchased from Amersham Pharmacia Biotech.
2.1-2 Enzymes, chemicals, equipments, and reagents
(1) Enzymes: All restriction enzymes were purchased from New England BioLabs lnc.
The pfu DNA polymerase was purchased from Stratagene. The T4 DNA ligase was purchased from Promega. All enzymes were used according to the recommended protocols.
(2) Chemicals: The following section lists the chemicals utilized in this study. The manufacturers were included in the square bracket. Acetic acid [Merck], Acrylamide [GE Healthcare], Agarose [USB], APS [GE Healthcare], α-Ala-L-His [Sigma],
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β-Asp-L-His [Sigma], Bestatin [MP Biomedicals], BactoTM Agar [DIFCO], L-carnosine [ICN Biomedicals, Inc.], Citric acid [Sigma], Coomassie® Brilliant blue R250 [Merck], Catechol [Alfa Aeser], Deoxyribonucleotide triphosphate (dNTP) 100mM Solutions [GE Healthcare], DNA 10kb Ladder [BioBasic Inc.], L-Dopa [Sigma], Dopamine [Sigma], Ethanol (95% and 99%) [Merck], GABA-His [Sigma], Glycine [Merck], Gly-Gly-His [Sigma], Gly-His-Gly [Sigma], Gly-His [Sigma], Glycerol [Merck], L-Histidine [Sigma], His-His [Bachem], His-Val [Bachem], His-Ile [Bachem], L-homocarnosine [Sigma], Hydrogen chloride [Merck], Ile-His [Bachem], Imidazole [USB], IPTG [GeneMark, Taiwan], Kanamycin sulfate [USB], LB Broth (Miller) [DIFCO], Leu-His [Bachem], Methanol [Merck], Oligonucletide Primers [BioBasic Inc.], Potassium chloride [Merck], Potassium diphosphate [Merck], Ser-His [Bachem], Sodium chloride [AMRESCO], Sodium hydroxide [Merck], SYBR® Green I [Roche], TEMED [GE Healthcare], Tris base [Merck], Tyr-His [Bachem], Val-His [Bachem], X-gal [GeneMerck, Taiwan], Yeast Extra [DIFCO].
(3) Kits: The following experimental kits were used in this study. BCA Protein Assay Reagent and Albumin Standard [PIERCE], GFXTM PCR DNA and Gel Band purification Kit [GE Healthcare], Plasmid Miniprep Purification Kit [GeneMark], QIAamp DNA Mini Kit [Qiagen], TOPO TA Cloning® Kit [Invitrogen], Plasmid Miniprep Purification Kit [GeneMark].
(4) Equipments: The following instruments were used in this thesis: ABI PRISM® 3100 Genetic Analyzer [Applied Biosystems], Amicon® Ultra [Millipore], Centrifuges 5415R [eppendorf], Electrophoresis Power Supply EPS 301 [GE Healthcare], Fluoroskan Ascent FL Microplate Reader [Thermo], GeneAmp® PCR System 9700 Thermal Cycler [Applied Biosystems], High-Performance Liquid Chromatography (HPLC) [Agilent], Kodak Electrophoresis Documentation an Analysis System 120 [Kodak], Mighty Small
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II for 8×7 cm gels electrophoresis instruments [GE Heathcare], Millex®-GS 0.22 μm Filter Unit [Millipore], Millex®-GS 0.45 μm Filter Unit [Millipore], Multiskan Ascent Microplate Reader [Thermo], SteritopTM 0.22μm Filter Unit [Millipore], Ultrasonic Processor VCX 500/750 [Sonics], UV-visible spectrometer [Amersham Biosciences].
(5) Reagents
Ampicillin stock solution (100mg/mL):
Dissolve 1 g Ampicillin sulfate in 10 mL double distilled water (ddH2O). Filter it through a 0.22 μm pore size filter and stock it at -20°C.
Destain buffer I:
Mix 400 mL methanol, 100 mL acetic acid and distilled water (dH2O) to 1 L. Store at room temperature.
Destain buffer II:
Mix 50 mL methanol, 120 mL acetic acid and dH2O to 1 L. Store at room temperature.
6 X DNA loading dye:
0.25% Bromophenol blue and 30% glycerol in ddH2O. Store at -20 °C.
IPTG stock solution:
Dissolve 4.086 g IPTG in 10 mL ddH2O. Filter through 0.22 μm pore size filter and store at -20 °C.
Kanamycin stock solution:
Dissolve 250 mg kanamycin sulfate in 10 mL ddH2O. Filter through 0.22 μm pore size filter and store at -20 °C.
LB medium:
25 g LB Broth was dissolved in 1 L ddH2O and sterilized.
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LB plate:
25 g LB Broth and 20 g BactoTM Agar was dissolved in 1 L ddH2O and sterilized. The sterile solution was poured and dispersed in Petri dishes before coagulation.
OPA reagent (for enzymatic activity assay):
Dissolve 50 mg OPA in 5 mL methanol first and then mix with 20 mL borate buffer. The borate buffer was mixed by 0.2 M boric acid (dissolved in 0.2 M potassium chloride solution) and 0.2 M sodium hydroxide solution (50 : 50, v/v).
10 X SYBR Green solution:
10,000 X SYBR® Green I was diluted to 10 X with DMSO. Store it under darkness.
50 X TAE buffer:
Add 242 g Tris-base, 57.1 mL Acetic acid, and 0.5 M EDTA into 800 mL ddH2O. Adjust the total volume into 1 L and pH value into pH 8.5. Store it at room temperature. Dilute the concentration to 1 X with ddH2O. Adjust the pH value to 7.5~7.8 before utlization.
X-gal stock solution:
Dissolve 400 mg X-gal in 10 mL dimethylformamide (DMF) and store in the darkness at -20 °C
2.2 Experimental methods
2.2-1 Site-directed mutagenesis of V. alginolyticus pepD
Site-directed mutagenesis of the V. alginolyticus pepD gene was carried out using the Stratagene QuikChange site-directed mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA). Mutagenesis primers were designed for use with the pET-28a(+)-pepD plasmid (wild-type) template (Appendix 1). The PCR reaction was carried out via the nonstrand-displacing action of pfuTurbo DNA polymerase to extend and incorporate the
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mutagenic primers, resulting in the nicked circular strands. The PCR products with wild-type and mutant plasmids were incubated with Dpn I for 4 hrs at 37 °C to selectively digest the methylated non-mutated parental wild-type plasmids. After Dpn I digestion, the mutant plasmids were transformed into E. coli XL-1 Blue competent cells, with selection for kanamycin resistance. Mutations were confirmed by restriction enzymes and DNA sequencing. The recombinant mutant plasmids were transformed into E. coli BL21(DE3) competent cells for expression of the mutated PepD proteins.
2.2-2 Construction of the truncated V. alginolyticus pepD catalytic domain gene
The truncated V. alginolyticus PepD catalytic domain gene (pepDCAT) was composed of the pepD gene sequence of nucleotides 1-558 and 1203-1470. An 826-bp fragment, which included 558 bp of the 5’-end and 268 bp of the 3’-end of the pepD gene, was amplified by PCR using the following primer pairs: CYC-PepD-BamHI-1 (sense primer, 5’-CGGGATCCGTGTCTGAGTTCCATTCTG-3’) and CYC-PepDcat-4 (antisense primer, 5’-TCCAGCCTGGTCCTGCACAACCCATGTACAC-3’) and by
CYC-PepDcat-3 (sense primer, 5’-TGGGTTGTGCAGGACCAGGCTGGAAACCAGATG-3’) and CYC-PepD-XhoI-2
(antisense primer, 5’-CGCTCGAGTTACGCCTTTTCAGGAATG-3’). The PCR product was subcloned into pET-28a(+)-pepDCAT, which was then transformed into E. coli BL21(DE3) pLysS competent cells for the production of the PepD catalytic domain protein (PepDCAT).
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2.2-3 Expression of the V. alginolyticus pepD gene, mutant pepD gene and truncated pepD gene in E. coli
The wild-type PepD, mutant PepD and PepDCAT proteins were produced in the same manner. Colonies grown on an LB plate were inoculated into LB broth supplemented with 50 μg/mL of kanamycin and grown at 37 °C until A600 of 0.5-0.6 was reached. At this point, protein production was induced by the addition of isopropyl thio-β-D-galactoside to a final concentration of 0.5 mM, and the culture was incubated at 37 °C for an additional 4 hrs before harvest. The cells were collected by centrifugation and then resuspended in 15 mL of 20 mM Tris-HCl (pH 7.0) buffer containing 0.5 M NaCl. The mixture was sonicated, and the cell debris was removed by centrifugation at 11,000 ×g for 30 min at 4 °C.
2.2-4 Portein purification of wild-type PepD, mutant PepD and PepDCAT
The wild-type PepD, mutant PepD and PepDCAT proteins were purified in the same protocol. The supernatant containing recombinant protein was loaded onto a Ni-SepharoseTM 6 Fast Flow column previously prepared by washing with 10 column volumes of buffer A (20 mM Tris-HCl, 0.5 M NaCl, pH 7.0) containing 20 mM imidazole. The protein-loaded column was first washed with 5 column volumes of buffer A + 20 mM imidazole, then with 5 column volumes of buffer A containing 70, 200, or 500 mM imidazole. Fractions of 1 mL each were collected, and the protein concentration in each fraction was determined using the PIERCE BCA Protein Assay Reagent with BSA as the standard. In addition, the eluted fractions were collected for SDS-PAGE analysis and enzymatic activity assay. By SDS-PAGE analysis, the high-purity eluted fractions were collected and dialyzed with 2 L of 50 mM Tris-HCl
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(pH 7.0) buffer for 2 hrs, followed by 3 L buffer for 8 hrs. After enzymatic activity assay, the purified recombinant proteins were stored at -80 °C for up to 6 months without loss of activity.
2.2-5 Protein concentration determination
The protein concentrations of purified proteins were measured using BCA Protein Assay Reagent. To each well of the F96 MicroWellTM plate was added a 20 μL sample mixed with 200 μL BCATM Working Reagents (BCATM Reagent A : BCATM Reagent B
= 50 : 1). The reactions were incubated at 37 °C for 30 min in the dark. The absorbances of samples were measured at 562 nm on a Multiskan Ascent Microplate Reader. 2 mg/mL bovine serum albumin (BSA) stock and successive dilutions (1.5, 1.0, 0.75, 0.5, 0.25, 0.125, 0.025 mg/mL) served as standards, following the same procedure described above.
2.2-6 SDS-PAGE and Native-PAGE analysis
After protein purification, gel electrophoresis was used to confirm for protein expression level, purity, and molecular weight. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% gels. Each 10 μL sample was mixed with 2 μL 5X SDS-PAGE sample buffer and incubated at 95ºC for 5 min to denature proteins. Electrophoresis was performed with 1X SDS-PAGE running buffer at 90 Volts for 30 min, followed by 120 Volts for 1.5 hrs. The SDS-PAGE gel was stained with a stain buffer containing Coomassie Brilliant blue R-250 for 30 min and destained with destain buffer I (methanol/acetic acid/water = 4:1:5, v/v/v) for 20 min, followed by destain buffer II (methanol/acetic acid/water = 1.2:0.05:8.75)
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overnight.
Native-PAGE was performed to examine the native form of PepD. The purified and dialyzed protein fractions were separated by Native-PAGE on 7.5% gels. The experimental steps were similar to SDS-PAGE analysis, except that the gel contained no SDS and there was no denaturing treatment. Each 10 μL sample was mixed with 2 μL 5X Native-PAGE sample buffer, and immediately followed by an iced 1X Native-PAGE running buffer at 90 Volts for 3 hrs in a 4ºC circulating water bath. The proteins were stained and destained in the same way as for SDS-PAGE analysis.
2.2-7 Enzymatic activity assay of PepD
PepD activity assay was according to a method described by Teufel et al15. on the basis of measurement of histidine derived with o-phthaldialdehyde (OPA) (Fig. 2.1). The reaction was initiated by addition of substrate and stopped by adding trichloroacetic acid (TCA) after 15 min incubation at 37 C. Histidine was produced from the substrate hydrolyzing by the enzyme. Then, fluorescence of OPA-derived L-histidine was measured using Fluoroskan Ascent FL (Exc: 355 nm and Em: 460 nm). The reaction with L-histidine and L-carnosine only solution were treated in the same way to serve as the positive and negative control, respectively. All reactions were carried out in triplicate.
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Figure 2.1. Formation of a Schiff base by L-histidine and o-phthalaldehyde.
2.2-8 Enzyme kinetics
For determination of Vmax, Km, and kcat of V. alginolyticus PepD wild-type and mutant proteins, the method described by Csámpai et al. was slightly modified for use with High Performance Liquid Chromatography (HPLC) and fluorescence detection. A system consisting of an Agilent 1100 Series Quaternary pump, Autosampler, Fluorescence Detector and Inertsil ODS-3 (7 μm, 7.6 mm×250 mm) column was used.
The eluent system consisted of two components: eluent A was 0.05 M sodium acetate at pH 7.2, while eluent B was prepared from 0.1 M sodium acetate–acetonitrile–methanol (46:44:10, v/v/v) (titrated with glacial acetic acid or 1 M sodium hydroxide to pH 7.2).
The gradient program was as described in Table 2.1. The fluent flow-rate was 0.8 mL/min at 30 ºC.
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Table 2.1: The fluent gradient program
Step Time (min) A (%) B (%) 1 0 100 0 2 5 50 50 3 15 25 75 4 20 0 100
Different concentrations of L-carnosine (0.25, 0.5, 1, 1.5, 2, 5, and 10 mM) were added as to a nanomolar concentration of enzyme solution in 200 μL at pH 7.0 for 20 min at 37 °C. The liberated histidine was derivatized with 100 μL OPA reagent for 5 min at 37 °C, and the fluorescence was detected as described previously. Fluorescence of the histidine with derivatived OPA was measured by FLD (λExc: 355 nm and λEm: 460 nm).
Various concentrations of L-histidine solution derivatived with OPA reagent were detected, using the method described above, to serve as standards. The data collected were applied to the Lineweaver-Burk equation. The kcat/Km values reflect values assuming 100% activity of the enzyme preparation.
2.2-9 Circular dichroism (CD) spectroscopy
The secondary structure of the wild-type and the mutant PepD proteins were confirmed by monitoring CD spectra. The protein sample concentration was 0.2 mg/mL in 50 mM Tris-HCl, pH 7.0 buffer. The CD spectra were recorded every 1 nm between 200 to 300 nm wavelength used a quartz cuvette of 1 mm path-length in a Jasco J-715 spectropolarimeter, Only 50 mM Tris-HCl, pH 7.0 buffer was as the control. The results were scanned 4 times and averaged. Converted the data into mean residue ellipticity (MRE) by using the equation : ［θ］MRE = (MRW × θobs/c × d). θobs is the observed ellipticity (in millidegrees) at the respective wavelength, MRW is the mean residue of
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the enzyme (MRW = M/n, M = 53548.8 g/mole, n = 490 amino acid residues), d is the cuvette path-length in cm, and c is the protein concentration in mg/mL.
2.2-10 Analytical sedimentation velocity ultracentrifugation
Sedimentation velocity is an analytical ultracentrifugation (AUC) method that measures the molecular moved rate for providing both the molecular mass and the shape of molecules. This technique can distinguish the native state of the protein in either a monomer, dimmer, or even tetramer form. The data were evaluated according to the g*(s) method developed by Walter Stafford. Since the g*(s) analysis yields both the sedimentation coefficient s from the peak of the curve, the apparent molecular weight can also be determined. Depending on the application and optical system, the protein concentration ranging 0.1 mg/mL to 0.5 mg/mL was used and the sample volume was about 500 μL. Sample was equilibrated with 20 mM Tris-HCl pH 7.0 buffer and this equilibrated buffer was used as another reference control into the reference sector. The sedimentation velocity analysis was performed at National Tsing Hua University.
2.2-11 Crystallization and data collection of PepD crystals
Crystallization of PepD was performed at 291 K by the hanging-drop vapor-diffusion method against a reservoir solution containing PEG 400 (28%, v/v), 0.2 M CaCl2 and 0.1M Na-HEPES buffer (pH 7.5), Crystals of a diamond shape appeared within six months and grew to maximum dimensions of 0.3 × 0.2 × 0.1 mm3. The protein crystals were transferred to the cryo protectant solution containing glycerol (15%, v/v) prior to the X-ray diffraction experiment. Diffraction data were collected to 3.0 Å resolution on SPXF beamline BL13B1 at the National Synchrotron Radiation Research
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Center (NSRRC) in Taiwan and beamline BL12B2 at SPring-8 in Japan. The data were processed using the HKL2000 suite79. The redundancy independent merging R factor (Rr.i.m.) and the precision indicating merging R factor (Rp.i.m.) were calculated using the program RMERGE80, 81. The crystals belong to space group P65 with unit cell parameters a = 80.42 Å and c = 303.11 Å. The asymmetric unit contained two protein molecules, corresponding to a solvent content of 53.4%.
2.2-12 Structure determination and refinement
The structure was solved by molecular replacement with MOLREP (CCP4) using the structure of Xaa-His dipeptidase from Haemophilus somnus 129PT (PDB code 2QYV) as the search model. The orientation of the lid domain was first located and fixed, subsequently leading to the determination of the relative position of the single catalytic domain. For structural refinement, the model was built using WinCOOT and refined using REFMAC5 (CCP4) to give the final Rwork = 0.231 and Rfree = 0.27482, respectively. The Ramachandran results were determined using MOLPROBITY, and the percentage of residues in favored, allowed, and disallowed were 94.5, 98.6, and 1.4%, respectively83. The structure found to have good stereochemistry was fully defined from Glu3 to Glu488, with all main chain angles in the most favorable or generally allowed regions84. All figures were produced using PyMOL. The atomic coordinates and structure factor data have been deposited in the Protein Data Bank (www.rcsb.org): PDB ID codes 3MRU.
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2.2-13 Substitution of zinc ions by copper ions to form CuCu-PepD
PepD protein first was dialyzed overnight with 50 mM Tris-HCl buffer at pH 7.5 containing 200 mM NaCl and 10 mM EDTA to remove divalent zin ions (apo-PepD).
The apo-PepD was dialyzed twice with the same buffer but without EDTA and exchanged with 50 mM Tris-HCl buffer at pH7.5 before adding 2mM CuCl2. After dialyzing overnight, the pooled CuCu-PepD protein then were dialyzed with a 50 mM Tris buffer (pH7.0) and stored at -20 °C.
2.2-14 Oxidative activity assay of CuCu-PepD
Oxidation of various catechol derivatives by CuCu-PepD were determined via the measurement of the products of o-quinone moiety (Fig. 2.2A). The oxidative products of dopamine, L-dopa, epinephrine and norepinephrine, which would be tautomerized to from the aminechrome derivertives (Fig. 2.2B) were detected at λmax: 480 nm, 475 nm, 475 nm and 490 nm, respectively.
Figure 2.2. Enzyme function of catechol oxidase. (A) Oxidation of catechol derivatives by catechol oxidase to form o-quinone moieties. (B) Overall reaction catalyzed by catechol oxidase with the substrate L-dopa. L-dopa undergoes oxidation to dopaquinone. Tautomerization of the dopaquinone ring by intramolecular nuclerophilic attck results in the formation of dopachrome.
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100 μL CuCu-PepD protein (1 mg/mL) and 900 μL 50 mM Tris buffer at pH 7.0 reacted with 2 mM substrates (catechol derivatives) at room temperature over 30 min.
The reactions containing only buffer with the substrates as negative controls. The absorbances of products were measured at the individual absorption wavelengths on a UV-visible spectrometer. All reactions were carried out in triplicate.
2.2-15 Kinetic analysis of CuCu-PepD for oxidative activity
For determination of Vmax, Km, and kcat of the CuCu-PepD, the oxidation kinetics were performed by the method described by Chen et al.85. The activity were determined at room temperature by following the increasing absorbance (dopamine: 480 nm; L-dopa:
475 nm; epinephrine: 475 nm; norepinephrine: 490 nm) accompanying the oxidation of the substrate with the molar absorption coefficient (dopamine: 3300 M-1cm-1; L-dopa:
3700 M-1cm-1; epinephrine: 4020 M-1cm-1; norepinephrine: 3580 M-1cm-1). Therefore, substitution of absorbance value and molar absorption coefficient into the equation, ΔA
= ε × b × Δc, where A refers to absorbance change per minute (ΔA/min), b is the light path length through the cuvette (1 cm), ε is the molar absorption coefficient for the product, and c is the change of concentration of product. The assay system was 1 mL containing 50 mM Tris buffer at pH 7.0, 0.1-5 mM substrate, and 0.925 μM CuCu-PepD at room temperature for 20min. For each substrate prepare a Michaelis-Menten curve (μmolar product/min versus mmolar substrate) and a Lineweaver-Burk plot. Absorption and kinetic measurements were carried out using a UV-visible spectrometer.
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2.2-16 Protein-ligand docking of CuCu-PepD for catechol derivatives
Docking is one of the useful tool to investigate the interaction between protein and ligand, protein and protein, and protein and DNA. Here, protein-ligand docking was carried out by the docking program – GOLD which was applied from National Center for High-Performance Computing (NCHC). We used the crystal structure of PepD (PDB code: 3MRU) as template, and the coordinates of ligands were download from PubChem.
Firstly, the PepD template was hydrogenation. Secondly, the ligand was defined around the Zn2 within 10 Å. Then, “restrict atom selection to solvent-accessible surface” and
“force all H bond donors/acceptors to be treated as solvent accessible” were selected.
Scoring function was selected for “ChemScore”, and parameter file was selected for
“kinase.params”. The program could be early termination, and used the internal ligand energy offset. After running the docking program, the protein-ligand binding states were ranked according to more stable energy. The interactions between proteins and ligands were shown by Pymol program.
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CHAPTER 3
Results and Discussion
3.1 Expression, purification, and crystallization of V. alginolyticus PepD, and X-ray data collection of the PepD crystals
The V. alginolyticus PepD protein was overexpressed in E. coli BL21(DE3)pLysS
The V. alginolyticus PepD protein was overexpressed in E. coli BL21(DE3)pLysS