1. Mutation, Isolation and Kinetic Characterization of S287W using pET28a-AmpC 𝜷-Lactamase
Abstract
𝛽-lactamase is the most common type of antibacterial resistance. A more clear understanding of the
biochemical mechanisms in the hydrolysis of 𝛽-lactam antibiotics can benefit the treatment of 𝛽-
lactamase producing bacteria. By using site directed mutagenesis and recombinant expression, we can
change specific residues in a protein to determine the impact on substrate catalytic efficiency. We
have mutated Serine 287 to Tryptophan and compared to the kinetic character of native AmpC. We
had determined that the mutation correlates with a decreased catalytic efficiency.
(A) (B)
Fig. 1: (A) Overall structure of AmpC from E. coli HKY28, with α-Helicies red, β-sheets green and
loops cyan.1 (B) Hydrolysis mechanism of β-lactam antibiotic cephalosporin, resulting in harmless bi-
products.3
Introduction
• A 𝛽-lactam antibiotic (Fig. 2 B – Cephalosporin) is characterized by a 𝛽-lactam, which is an amide
within a four membered ring.
• The first 𝛽-lactam antibiotic was accidently discovered by Alexander Fleming in 1928, Penicillin,
from the fungi genus penicillium.2
• In the 1950’s however, the future of 𝛽-lactam antibiotics was thought to be coming to an end due to
the appearance of resistant S. aureus had been discovered in hospital environments.2
• Through side chain modification, the 𝛽-lactam antibiotic were once again effective, for a period of
time.2
• Today, the fight against anti-bacrterial resistance continues.
(A) (B)
Fig. 2: (A) Active site residues of a native Class C 𝛽-lactamase. (B) S287W mutant.
• 𝛽-lactamase is an enzyme which has an affinity for 𝛽-lactam antibiotics.
• Class A, C, and D 𝛽-lactamase use catalytic serine residues, which attack at the carbonyl carbon of
the 𝛽-lactam ring, with the difference being in substrate promiscuity and affinity.
• Class B uses a Zn2+ ion to hydrolyze a 𝛽-lactam ring.
• We want to determine the effect of residue 287 on the structure of AmpC.
(A) (B)
Fig. 3: (A) Sequencing chromatography of mutant AmpC using Sanger Sequencing, indicative of
mutation at residue 287. (B) pET28a-AmpC plasmid map using pDRAW.
Results
(A) (B)
(C)
(D)
Fig. 4: (A) SDS-PAGE, (B) Western Blot, T represents time intervals (in minutes) of samples taken
throughout the expression process of mutant AmpC protein. The lanes P (pooled), F (flow), W
(wash), and L (lysate) throughout the affinity chromatography. Western Blot from SDS-PAGE
transferred to nitrocellulose membrane blocked with dry lactate, and identified with primary anti-T7
tag. (C) Agarose Gel Electrophoresis standard curve molecular weight standard, showing log (#bp) vs
migration distance in cm. Some mutant AmpC was lost in the wash and flow of the affinity
chromatography. (D) Agarose Gel Electrophoresis of restriction endonuclease digestion.
• DNA Purification and Quantitation –
We had first isolated the pET28a-AmpC plasmid utilizing alkaline lysis. We used the Wizard® Plus
SV Minipreps DNA Purification System to isolate the plasmid DNA. Afterwards, DNA concentration
was determined using Nanovue, indicating a 1.86 𝐴260/𝐴280 and concentration of 0.0749
µg
µ 𝐿
.
• Restriction Endonuclease –
The restriction digest we performed used the restriction enzymes XbaI and NruI. In order to confirm
the correct plasmid was isolated, we had performed an agarose gel electrophoresis (Fig. 4 D). The
bands for the double digest on the gel electrophoresis aren’t ideal when comparing to the plasmid
map; the lowest band is very dim, and there is a band high up, indicating undigested plasmid.
Predicted # bp = 10
𝑀𝑖𝑔𝑟𝑎𝑡𝑖𝑜𝑛 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒−33.3
−6.916 = 10(
11.8−33.3
−6.916
)
= 1162
• Transformation –
We had used CaCl2-competent BL21(DE3) E. coli species expressing the T7 RNA Polymerase
system, the bacteria will uptake the plasmid DNA.
(A) (B)
Fig. 5: CENTAAssay on the activity of (A) S287W mutant (1:10 dilution) and (B) WT AmpC
uninhibited (red and green respectively) and inhibited (blue and violet respectively) with Cefataxime.
With the dilution factor taken into consideration, the mutant strain has about half the vmax of the native
strain when uninhibited.
• Overexpression and Purification of AmpC –
Isopropyl-β-D-Thiogalactopyranoside (IPTG) is used to inactivate the repressor of the lacZ operator,
allowing transcription. We had purified AmpC using affinity chromatography. We had set up the
column to have a Ni2+ ligand bind to the hexa-histidine tag, and dissociate from Ni2+ using imidazole.
• SDS-PAGE and Western Blot –
We had performed two SDS-PAGE samples, one for Coomassie staining (Fig. 4 A) and one for
Western Blot analysis (Fig. 4 B). The quantity of mutant AmpC increases as time proceeds after the
addition of IPTG. The concentration of mutant AmpC increases following the affinity
chromatography, in the pooled sample. The bands nearby the 37kDa are expectantly mutant AmpC.
• Kinetics –
We had performed cephalothin nitrothiobenzoic acid (CENTA) assays to assess the activity of our
mutant and native enzyme with and without Cefataxime inhibition.
kcat = 𝑣 𝑚𝑎𝑥 ÷ 𝐸𝑛𝑧𝑦𝑚𝑒 = 5.25 × 10−4 𝑀
𝑚𝑖𝑛
÷ 0.0110𝑀 = 0.0477𝑚𝑖𝑛−1
ki =
𝑘 𝑀×[𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒]
𝑘 𝑀𝑎𝑝𝑝−𝑘 𝑀
=
8.18×10−2 𝑀×6.67×10−4 𝑀
3.48×10−1 𝑀−8.18×10−2 𝑀
= 206µ𝑀
Conclusion
• Based on the agarose gel electrophoresis (Fig. 4 D), the plasmid fragments match up with the
predicted fragment sizes.
• With the data collected from the agarose gel electrophoresis, Western Blot (Fig. 4 B) and CENTA
activity assay (Fig. 5), we suggest the evidence points to the expression AmpC β-lactamase.
• The approximate molecular weight is about 37kDa, the antibody from the Western Blot had bound
to the T7 tag, and the sample had activity in the CENTAAssay.
• Compared to native AmpC, the mutated S287W clearly has a diminished catalytic efficiency for the
substrate CENTA.
References and Acknowledgements
1. Yamaguchi, Yoshihiro, Genta Sato, Yuriko Yamagata, Yohei Doi, Jun-ichi Wachino, Yoshichika Arakawa, Koki Matsuda, and Hiromasa Kurosaki.
"Structure of AmpC β-lactamase (AmpCD) from an Escherichia Coli Clinical Isolate with a Tripeptide Deletion (Gly286-Ser287-Asp288) in the
H10 Helix." Acta Crystallographica Section F. International Union of Crystallography. Web. 16 Apr. 2015.
2. Demain, Arnold L., and Richard P. Elander. "The β-lactam Antibiotics: Past, Present, and Future." Antonie Van Leeuwenhoek 75.1-2 (1999): 5-19.
Print.
3. Chen, Yu, George Minasov, Tomer A. Roth, Fabio Prati, and Brian K. Shoichet. "The Deacylation Mechanism of AmpC β--Lactamase at Ultrahigh
Resolution." JACS 13 Oct. 2005: 2970-976. Print.
Alexander James Ward, David Buck, Dr. Powers, R. A.
Department of Biochemistry and Cell and Molecular Biology
Grand Valley State University, Allendale, MI 49401
0.00E+00
5.00E-06
1.00E-05
1.50E-05
2.00E-05
2.50E-05
0 0.2 0.4 0.6
V0(M/min)
[CENTA] (mM)
0
0.0001
0.0002
0.0003
0.0004
0.0005
0 0.1 0.2 0.3 0.4 0.5 0.6
V0(M/min)
[CENTA] (mM)
PCR+ PCR- MW XbaI NruI
Double
Digest
Gly
286
Trp
287
Asp
288
Table 1: Agarose Gel Electrophoresis Fragments
Type #bp log (#bp) Migration Distance (cm) Predicted # bp
PCR+ ~1000 3.00 12.1 1162
XbaI 6419 3.81 7.04 6270
NruI 1293, 5781 3.11, 3.76 10.5, 7.98 1990, 4590
Double 3744, 1931, 744 3.57, 3.29, 2.87 13.6, 10.4, 8.43 3940, 2040, 710
T0 MWT15 T30 T45 T60 P F W L
37kDa
25kDa
50kDa
MW T0 T15 T30 T45 T60
P F W L
Table 2: Kinetic Parameters
Type kM (M) vmax (M/min) kcat (min-1) ki (µM)
Native AmpC 8.18 × 10−2
5.25 × 10−4
4.79 × 10−2
206
Native AmpC
Inhibited
3.48 × 10−1 4.52 × 10−4 4.11 × 10−2 N/A
S287W 1.44 × 10−2 2.55 × 10−4 6.97 × 10−3 34
S287W Inhibited 9.92 × 10−2
2.01 × 10−5 5.49 × 10−4
N/A
Ser64
Lys315
Lys67
Tyr150
Ser287
Trp287
Lys67
Ser64
Tyr150
Lys315
y = -6.916x + 33.3
5
6
7
8
9
10
11
12
13
14
15
2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4
log(#bp)
Relative Migration Distance (cm)