Mutation, Isolation and Kinetic Characterization of S287W using pET28a-AmpC -Lactamase

Abstract-lactamase is the most common type of antibacterial resistance. A more clear understanding of the biochemical mechanisms in the hydrolysis of -lactam antibiotics can benefit the treatment of -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 -lactam antibiotic (Fig. 2 B – Cephalosporin) is characterized by a -lactam, which is an amide

within a four membered ring. • The first -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 -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 -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 -lactamase. (B) S287W mutant.

• -lactamase is an enzyme which has an affinity for -lactam antibiotics. • Class A, C, and D -lactamase use catalytic serine residues, which attack at the carbonyl carbon of

the -lactam ring, with the difference being in substrate promiscuity and affinity. • Class B uses a Zn2+ ion to hydrolyze a -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 and concentration of 0.0749.

• 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 =

• 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: CENTA Assay 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 = ki =

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 CENTA Assay. • Compared to native AmpC, the mutated S287W clearly has a diminished catalytic efficiency for

the substrate CENTA.

References and Acknowledgements1. 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 0.1 0.2 0.3 0.4 0.5 0.60.00E+00

5.00E-06

1.00E-05

1.50E-05

2.00E-05

2.50E-05

[CENTA] (mM)

V0 (M

/min

)

0 0.1 0.2 0.3 0.4 0.5 0.60

0.0001

0.0002

0.0003

0.0004

0.0005

[CENTA] (mM)

V0 (M

/min

)

PCR+ PCR- MW XbaI NruI Double Digest

Gly 286

Trp 287

Asp 288

Table 1: Agarose Gel Electrophoresis FragmentsType #bp log (#bp) Migration Distance (cm) Predicted # bpPCR+ ~1000 3.00 12.1 1162XbaI 6419 3.81 7.04 6270NruI 1293, 5781 3.11, 3.76 10.5, 7.98 1990, 4590Double 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 T60P F W L

Table 2: Kinetic ParametersType kM (M) vmax (M/min) kcat (min-1) ki (µM)Native AmpC 206Native AmpC Inhibited

N/A

S287W 34S287W Inhibited N/A

Ser64

Lys315

Lys67

Tyr150

Ser287

Trp287

Lys67

Ser64Tyr150

Lys315

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 45

6

7

8

9

10

11

12

13

14

15

f(x) = − 6.91601161600054 x + 33.3004604875694

Relative Migration Distance (cm)

log(

#bp)