Based on the results in this study, two putative active site residues which were apparently involved in the catalysis of PepD were first identified. The saturated mutagenesis on other putative metal binding and catalytic sites will be continuous analyzed to clarify their roles in the peptidase activity of PepD. Moreover, several residues outside of the catalytic domain of enzymes in M20 family were considered to be involved in the substrate binding and catalysis in recent report. As described on section 1.9, the respective His269 from the lid domain of L.
elbrueckii PepV was associated with Zn 1 for forming an oxyanion binding hole bound to e carbonyl oxygen of Glu 153 and led to a tetrahedral intermediate. This residue functioned r the stabilization of the transition state and its corresponding results to His229, His223, and His206 in the small domains of the dimeric homologs CPG2, PepT, and hAcy1 in M20 family were also be examined. The mutagenesis study on H206 of hAcyl in 200326 indicated at the conserved histidine in the dimerization domain of dimeric Acy1/M20 family
zymes contributes in trans to the active site. Therefore, several polar or aromatic residues outside of the putative active site of PepD should also be futher investigated by site-directed mutagenesis in the future.
PepD was identified as a member of metallopeptidases which required metal ion for its catalytic activity. In these peptidases, the metal ion is usually zinc but sometimes cobalt, manganese, nickel or copper. However, recent studies showed that different metal ion with rious concentration could inhibit or increase the enzyme activity.42, 64 The functional roles r the different metal centers as well as the activation mechanism due to lose metal ions are still unclear. Consequently, the metal selectivity and inhibition/activation mechanism will be valuable investigated in the future.
d th fo
th en
va fo
As the biological function of bacterial pepD is less understanding, the gene knock-out study on V. alginolyticus pepD followed by a series of biochemical or morphology analysis will provide more informations about its role in prokaryotes. At the same time, since PepD affects the bacterial biofilm formation, biofilm assay should also be performed and compared with both V. alginolyticus wild-type and pepD knockout strain.
With absence of the structure in neither peptidase family M20 nor similar peptidase, crystallization on the V. alginolyticus PepD was needed. Furthermore, the crystal structure of the wild-type and mutant proteins combined with the mutagenesis analysis data could provide an insight into the catalytic mechanism of bacterial aminoacylhistidine dipeptidase.
Up to date, we have obtained the crystal of wild-type PepD with undesirable resolution.
Therefore, the proceeding effort is still needed to modify the crystallization condition in order to improve the quality of crystal for further structure determination.
Cha
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Appendix 1
Primers used in this thesis
Sequencing and Expression
F1 (sense) 5’-GTGTCTGAGTTCCATTC-3’ (1-17)a
F2 (sense) 5’-TGGGCGACAGAGCAAGG-3’ (127-143)
TCTGGCGCTTACCCAGG R1 (antisense) 3’-AAGGACTTTTCCGCATT-5’
F3 (sense) 5’- -3’ (1189-1205)
(1457-1473) R2 (antisense) 3’-CGTAACTTGCGAACAGG-5’ (979-995) R3 (antisense) 3’-GTGACTAGTGCTGAAGT-5’ (270-286) N1 (sense) 5’-CGCGGATCCCATATGGTGTCTGAGTTCCATTC-3’ (NdeI)b
Mutagenesis
D119X-1 5’-CGCTCGGGGCANNNAACGGCATCGGCATGGC-3’ (AvaI) D119X-2 5’-GCCATGCCGATGCCGTTNNNTGCCCCGAGCG-3’ (AvaI) E149X-1 5’-CTGACAATTGATNNNGAAGCAGGCATGACAGG-3’ (MfeI) E149X-2 5’-CCTGTCATGCCTGCTTCNNNATCAATTGTCAG-3’ (MfeI)
Appendix 2
The restriction enzyme used in expresson vectors check and the expected size of excised f
sed Excised frangm ragment.
Plasmid R.E.a u ent
pET-pepD-WT AvaI -b
pET-pepD-D119X AvaI 1.2 kbp.
pET-pepD-WT PvuI -b
pET-pepD-E149A MfeI/NotI 1.2 kbp.
a R.E.= restriction enzymes.
N fragment was excised.
b No expected D A
Appendix 3
Experimental Materials
l strains, plasmids, animal, and cell
scherichia coli BL21(DE3)pLysS (Novagen)Escherichia coli XL1-Blue (Novagen)
Vibrio algino 749 (FIRDI, Taiw pCR®2.1-TO
pET-28a(+) (
Female BAL cience Co n)
M
Chemicals and Reagents
cetic acid (Merck) ovine Calf Serum (HyClone) romophenol blue (USB)L-carnosine (ICN Biomedicals, Inc.) Sigma)
oomassie® Brilliant blue R 250 (Merck) Dimethylformamide (Merck)
Bacteria
Elyticus ATCC 17 an)
PO (Invitrogen) Novagen)
B/c mice (National S uncil, Taiwa ouse myeloma cell line FO (FIRDI, Taiwan)
A
Dimethyl sulfoxide (MP Biomedicals) Dodecyl sulfate sodium salt (M
ulbecco’s Modified Eagle Medium (Gibco) dNTP Set, 100 mM Solutions (GE Healthcare) Ethylenediamine-tetraacetic acid (Merck) GABA-His (Sigma)
L-glutamine solution 100X, 200m Glycerol (Merck)
HT Supplement (100X), liquid (GIBCO) Hydrogen chloride (Merck)
Ile-His (Bachem) Imidazole (USB)
Kanamycin sulfate (USB) LB Broth, Miller (DIFCO) Leu-His (Bachem)
2-mercaptoethanol (Merck) Methanol (Merck)
N,N’-methylene-bis-acrylamide (Sigma) Ni-NTA His-Band® Resin (Novagen)
Penicillin-Streptomycin Solution 100X (biowest) erck)
hate (Merck) ate (Merck)
., Taiwan)
es (New England Biolabs)
RESCO)
Kits
BCA Protein Assay Reagent and Albumin Standard (PIERCE)
ing Kit (Applied Biosystems) ication Kit (GE Healthcare)
ealthcare) gen)
en)
Kit (GeneMark)
fer II Pack (Applied Biosystems)
lyzer (Applied Biosystems) eckman Coulter)
eckman Coulter)
ase Scientific Glass, Inc.) orf)
l B401L (Firstek Scientific) Centrifuge 2100 (KUBOTA)
ath (Baxter) s (BIO-RAD)
ly EPS 301 (GE Healthcare) PSON® GT-7000 Scanner (EPSON)
96 MicroWellTM plate (black) (NUNC) BigDye® Terminator v3.1 Cycle Sequenc GFXTM PCR DNA and Gel Band Purif HMW Native Marker Kit (GE Healthcare) LMW-SDS Marker Kit (GE H
QIAamp DNA Mini Kit (Qia TOPO TA Cloning® Kit (Invitrog Plasmid Miniprep Purification rTth DNA polymerase, XL & XL Buf
Equipments
25 cm2 flask (NUNC)ABI PRISM® 3100 Genetic Ana AllegraTM 21R Centrifuge (B Avanti® J-E Centrifuge (B Blood Collecting Tubes (Ch Centrifuges 5415R (eppend Colling Circulator Bath Mode Compact Tabletop
Dri-Bath Type 17600 (Thermolyne) DurabathTM Water B
Econo-Pac Column
Electrophoresis Power Supp E
F
F96 MicroWellTM plate (clear) (NUNC)
s) Unit (GE Healthcare) Healthcare)
tion and Analysis System 120 (Kodak) ents (GE Healthcare)
ultiskan Ascent Microplate Reader (Thermo)
ubator Model S300R (Firstek Scientific) l RS-101 (Firstek Scientific)
-Jacketed Incubator (NuAire) Healthcare) Fisher Vortex Genie 2TM (Fisher Scientific)
Fluoroskan Ascent FL Microplate Reader (Thermo)
GeneAmp® PCR System 9700 Thermal Cycler (Applied Biosystem Hoefer® HE 33 Mini Horizontal Submarine
Hoefer® Mighty Small dual gel caster (GE Kodak Electrophoresis Documenta
Mighty Small II for 8×7 cm gels electrophoresis instrum Millex®-GS 0.22 μm Filter Unit (Millipore)
Millex®-HA 0.45 μm Filter Unit (Millipore) M
Orbital shaking inc Rocking Shacker Mode
SteritopTM 0.22 μm Filter Unit (Millipore) Ultrasonic Processor VCX 500/750 (Sonics) US AutoFlowTM NU 4000 Series CO2 Water
UV-Visible Spectrophotometer Ultrospec 3100 pro (GE
Solutions
Blocking buffer
5% non-fat milk in distilled water (dH2O).
Destain buffer I
Mix 400 mL methanol, 100 mL acetic acid and dH2O to 1 L. Store at room temperature (RT).
distilled water (dH2O) to 1 L. Store at RT.
distilled water (ddH2O). Store at -20℃.
e filter and store at
anamycin stock solution
issolve 250 mg kanamycin sulfate in 10 mL ddH2O. Filter through 0.22 μm pore size filter nd store at -20℃.
B medium
5 g LB Broth was dissolved in 1 L dH2O and sterilized.
Destain buffer II
Mix 50 mL methanol, 120 mL acetic acid and
6X DNA loading dye
0.25% bromophenol blue and 30% glycerol in double
IPTG stock solution
Dissolve 4.0863 g IPTG in 10 mL ddH2O. Filter through 0.22 μm pore siz -20℃.
K D a
L 2
LB plate
25 g LB Broth and 20 g BactoTM Agar was dissolved in 1 L dH2O and sterilized. The sterile ed and dispersed in petri dishes before it coagulates.
0X Native-PAGE running buffer
ycine and 30 g Tris base in 1 L dH2O and store at 4℃. Dilute to 1X with
sample buffer
H2O ere mixed and stored at -20℃.
issolve 50 mg OPA in 5 mL methanol first and then mix with 20 mL borate buffer. The d by 0.2 M boric acid (dissolved in 0.2 M potassium chloride solution)
or no longer than 9 days and prepared at least 90 min earlier before use.
2O and sterilize before use.
E running buffer
L dH2O and store at 4℃. Dilute to X with dH2O before use.
LB agar was pour
1
Dissolve 144 g gl dH2O before use.
5X Native-PAGE
8 mg bromophenol blue, 1.7 mL 0.5 M Tris-HCl, pH 6.8, 5 mL glycerol, and 4 mL d w
OPA reagent (for enzyme kinetics) D
borate buffer was mixe
and 0.2 M sodium hydroxide solution (50: 50, v/v). The OPA reagent was stored in darkness at 4℃ f
10X PBS buffer
Dissolve 13.7 g Na2HPO4, 3.5 g NaH2PO4, and 87.7 g NaCl in 1 L dH2O and store at RT.
Dilute to 1X with dH
10X SDS-PAG
Dissolve 144 g glycine, 30 g Tris base, and 10 g SDS in 1 1
5X SDS-PAGE sample buffer
red at -20℃.
2O to 1 L final volume. Filter through reused 0.22 μm pore size filter and ore at RT.
acid 57.1 mL, and 0.5 M EDTA in 1 L dH2O and adjust to H 8.5. Dilute to 1X with dH2O and adjust to pH 7.5-7.8 before use.
-gal stock solution
L dimethylformamide (DMF) and store in the darkness at 8 mg bromophenol blue, 1.7 mL 0.5 M Tris-HCl, pH 6.8, 0.5 mL 20% (w/v) SDS, 2 mL 2-mercaptoethanol, 5 mL glycerol, and 4 mL dH2O were mixed and sto
Stain buffer
Dissolve 1 g Coomassie Brilliant blue R-250 in 500 mL methanol first. Then add 100 mL acetic acid and dH
st
50X TAE buffer
Dissolve Tris base 242 g, acetic p
10X Western transfer buffer
Dissolve 144 g glycine, 30 g Tris base, and 10 g SDS in 1 L dH2O and store at 4℃. Dilute to 1X with dH2O before use.
X
Dissolve 400 mg X-gal in 10 m -20℃.