Site-Directed Mutagenesis and Purification of DNA for K99S Mutant of Aspartate Aminotransferase

Abstract

The objective of this study is to identify key residues in determining the substrate specificity of aminotransferases. This experiment in particular aims to evaluate if site-directed mutagenesis at one key residue (K99S) can eradicate the aspartate specificity of aspartate aminotransferase (TM1255) in Thermotoga maritima and induce alanine specificity. The site for mutation was chosen after structural alignment with the aspartate aminotransferase from Thermus thermophilus and sequence alignment with the wild type alanine aminotransferase from T. maritima. PIPE mutagenesis introduced the mutation into the wild type genome, and the gene for the protein of interest was transformed into HK100 competent E. Coli cells. Qiagen Miniprep was used to purify the DNA sample, and nanodrop quantitation determined concentration to be 64.2 ng/μL. With an A260/280 of 1.83 and an A260/230 of 1.61, the DNA sample is free of RNA contamination but may contain traces of aromatic organic compounds. Sequencing of the DNA shows successful mutation at the intended site with conservation of all other amino acids.

IntroductionAspartate aminotransferase, one of a class of aminotransferases, catalyzes the

transamination of aspartate to form glutamate and oxaloacetate as shown in Figure 1.1

Figure 1. Aspartate Transamination Reaction. Aspartate aminotransferase catalyzes the transfer of amino group from aspartate to -ketoglutarate to form glutamate and oxaloacetate. In the pharmaceutical industry, information on substrate binding is needed in order to

engineer drug inhibitors to prevent the binding of one aminotransferase while continuing to

allow all others.2 The regulation of aminotransferase has important implications for individuals

suffering from myocardial infarction.3 The purpose of this research is to identify key residues in

determining the substrate specificity of aminotransferases. Specifically, this investigation studies

the site-directed mutagenesis of one identified residue in an attempt to change the specificity of

aspartate aminotransferase to alanine.

Research into the dual substrate recognition of aminotransferases shows evidence that

substrate specificity involves a combination of the induced fit

and lock and key models.4 This suggests that substrate binding

is dependent on (1) the large scale network of hydrogen

bonding in the active site and (2) the hydrophobic/hydrophilic

nature of active site amino acid residues and their interactions

with the substrate.4 There are two methods to engineer these

active sites, de novo protein design and protein redesign.5

Currently, a better understanding of protein folding is needed

to successfully implement de novo protein design, but by

taking advantage of existing protein structures, protein function

can be altered by site-directed mutagenesis.5

The steps taken in obtaining DNA by site-directed

Figure 2. Steps of PIPE Mutagenesis mutagenesis are outlined in Figure 2. Polymerase Incomplete

and Purification.6 Primer Extension (PIPE) mutagenesis was chosen as a cheaper

Design primer with desired mutation

PCR of plasmid with mutagenic primers

Transform Top10 competent E. Coli cells

with PCR product

Culture individual colonies + purify using

Qiagen Miniprep kit

and faster method to target one site for mutation.6 PIPE mutagenesis involves two steps: PCR

amplification and cellular transformation.7 PCR amplification uses DNA polymerase to

synthesize complementary strands of DNA from a template and a primer for the desired

mutation.8 Exponential amplification of the intended sequence is achieved by repeated cycles of

denaturation, annealing, and elongation. The entire PCR mixture is then transformed into E.Coli

to repair and ligate ends in vivo, thereby creating a replicating plasmid.6 Qiagen Miniprep

purification was then used to isolate a pure DNA sample to use in growth of the mutant protein.

Quantitation by nanodrop provided necessary information on concentration and absorbance to

determine the amount of sample to send for sequencing and the success of purification.10 The

A260/280 ratio indicates any RNA contamination in the sample where pure DNA has an expected

A260/280 of 1.8 and RNA raises this ratio closer to 2.0.9 The A260/230 ratio reveals any organic

contamination such that aromatic compounds that absorb at 230 nm lower the ratio below 1.8.10

Nanodrop quantitation determined the exact concentration of the sample and

demonstrated an adequately pure DNA with small traces of aromatic contamination.

Furthermore, DNA and protein sequencing show successful mutation to the K99S mutant

conserving all other amino acids. Confirmation that the DNA was successfully mutated allows

further kinetic research can be done to test the substrate specificity of the K99S mutant of

aspartate aminotransferase that can be confidently grown from this sample.

Materials and Methods

The active site of aspartate aminotransferase was determined by structural alignment of

the protein of interest from Thermotoga maritima and Thermus thermophilus using the

educational version of PyMol v1.7.4. T. Thermophilus was chosen for structural comparison as

previous research shows a high number of functionally similar residues conserved in the active

site of aspartate aminotransferases in comparison to both prokaryotes and eukaryotes such as

E.coli.11 Active site residues were identified within four angstroms of the bound cofactor PLP.

Sequence alignment comparison of the active site using pBLAST between aspartate

aminotransferase and alanine aminotransferase was used to identify the best residue for

mutagenesis. Mutagenesis and docking of substrate was modeled in PyMol to assess plausibility

of a change in specificity.

PIPE Mutagenesis. PCR reactions were conducted using a master mix containing ~3

ng/μL template DNA, 10x Pfr turbo reaction buffer, 2.0 mM dNTPs, ddH2O, and Pfu Turbo

polymerase. Each PCR tube containing 5 μL primer and 40 μL master mix was loaded into a

BioRad C1000 Thermal Cycler along a temperature gradient from 48-64°C. Four PCR reactions

were run at annealing temperatures of 64°C, 60.7°C, 54.1°C, and 49.1°C. The thermocycler ran

reactions for 8 hours and 44 minutes according to the program described in Table 1.

Table 1. Thermocycler PCR ProgramPCR Reaction Step Temperature (°C) TimeInitial Denaturation

(activate polymerase)95 180 s

Subsequent Denaturation

95 30 s

Annealing 48-64 45 sElongation 68 14 minutes

Number of cycles 30 cyclesFinal Elongation 68 7 minutes

Hold 4 IndefiniteGel Electrophoresis. A 0.8% (v/w) agarose gel made in 1x TAE buffer and 1% Ethidium

Bromide was used to qualitatively determine the optimal annealing temperature used in

mutagenesis for DNA transformation. 10 μL of PIPE PCR reaction with 2 μL 6X Nucleic Acid

Loading Buffer was run in the gel at 120 volts for 30 minutes. The gel was visualized using a Gel

Doc EZ Imager, and a Promega 1 kb DNA Ladder Standard was used as a molecular weight

marker.

DNA Transformation. The sample with the best PCR annealing temperature as

determined by the gel results was mixed with HK100 competent E. coli cells for DNA

transformation. The mixture was incubated on ice for 20 minutes, at 42°C for 45 seconds, and on

ice again for 2 minutes before adding LB medium without antibiotics. The transformation tube

was incubated at 37°C for 45 min with shaking at 225 rpm. A 40 μL sample from the

transformation tube was spread onto an LB-Amp agar plate and incubated overnight at 37°C.

Mini-Prep. Mutant DNA was collected using a Qiagen Qiaprep Spin Miniprep kit. An

overnight culture was incubated in a culture tube at 37°C with shaking for 12-16 hours, and cells

were harvested by centrifugation in two 2 mL tubes. Each cell pellet was re-suspended in 125 μL

Buffer P1 and combined into one centrifuge tube. 250 μL Buffer P2 and 350 μL Buffer N3 were

added before centrifuging for 12 minutes at full speed. The supernatant was pipetted off the

pellet into a Qiagen column and spun for another minute. The flow through was discarded after

500 μL Buffer PB was added and spun for 1 minute, and the wash was discard from centrifuging

with 750 μL Buffer PE. A mixture of 40 μL dH20 was mixed and the supernatant was let sit for 1

minute in a new centrifuge tube before spinning for 1 minute at full speed. The flow through

containing DNA was retained for analysis.

Quantitation and Sequencing. Absorbance and concentration of the DNA sample was

determined using NanoDrop, and 10 μL of DNA sent for sequencing by GeneWiz. DNA

sequence was translated to protein sequence using ExPasy, and the success of the mutation was

determined by comparing the mutant to the wild type of 1o4s using pBLAST.

Results

The structural alignment of the protein of interest TM1255 and another aspartate

aminotransferase in Thermus Thermophilus shown in Figure 3 was used to determine conserved

active site residues within 4 angstroms of bound PLP.

Figure 3. Structural alignment of the wild type AspAT from Thermotoga maritima (blue) and another AspAT from Thermus thermophilus (orange). The POI from Thermotoga maritima (TM1255) and an aspartate aminotransferase from Thermus Thermophilus were structurally aligned in PyMol with bound PLP in green. Both proteins catalyze the transamination of aspartate, and conserved active site residues are shown with POI residues as the first number and the homolog as the second.

The sequence alignment of the active sites of aspartate aminotransferase (TM1255) and

alanine aminotransferase (TM1698) in T. maritima is shown in Figure 4. A stretch of residues

was identified as belonging to the same class of amino acids with one significant difference

between lysine in AspAT and serine in AlaAT at residue 99.

Figure 4. Sequence Alignment of Residues for AspAT (81-121), and AlaAT (82-122). The residues highlighted in light blue belong to the same classes of amino acids, and those highlighted in yellow are active site residues that differ significantly between each aminotransferase.

Mutagenesis of K99S in TM1255 was modeled in PyMol to compare the distance

between the mutant serine residue in AspAT and docked alanine and that of the wild type AlaAT

in Figure 5.

Figure 5. Comparison between the Mutant AspAT and AlaAT. A) The stick model of wild type AlaAT (yellow) for Thermotoga maritima docked with alanine substrate (orange) with the side chain (red) pointing towards the Ser-100 side chain (blue) on the AlaAT. The distance between the serine side chain and alanine of substrate is measured in angstroms. B) The stick model of mutant AspAT (K99S) for Thermotoga maritima docked with alanine substrate (orange) with the side chain (red) pointing towards the Ser-99 side chain (blue) on the AspAT. The distance between the serine side chain and alanine of substrate is measured in angstroms.

Figure 6 displays the results of gel electrophoresis after PCR. With a plasmid of 6 kbp

and a mutation with approximately 5.3 kbp, the protein would be located between 5,000 and

6,000 bp. According to these results, the sample using an annealing temperature of 64 °C was

used for DNA transformation.

Figure 6. Gel electrophoresis of the PCR reaction of the K99S mutant in an E. Coli Plasmid at four different Annealing Temperatures. 1: Promega 1 kb DNA ladder Standards; 2: annealing at 64 °C; 3: annealing at 60.7 °C; 4: annealing at 54.1 °C; 5: annealing at 49.1 °C. The bands in lanes 2-5 appear between 6,000 and 5,000 and correspond to the plasmid with the proposed mutation, which should be approximately 5.3 kb.

Following DNA transformation, cell culture plates in Figure 7 show many colonies.

Table 2 summarizes the results of quantitation by NanoDrop. DNA sequencing results returned

from GeneWiz were translated to protein sequence and compared to the wild type sequence in

Figure 8.

Figure 7. Transformation plates of PCR reaction mixture of TM1255 of the mutant K99S at the annealing temperature of 64 °C. Many colonies were visible indicating a good

transformation.Figure 8. The protein sequence alignment obtained through NCBI Blast: Protein Sequence. The query sequence is the mutant aspartate aminotransferase K99S, while the subject sequence is the wild-type sequence of aspartate aminotransferase. The highlighted residue 99 was successfully

mutated from a lysine (Wild-Type) to a serine (mutant).

Discussion

The proposed K99S mutation is plausible for shifting substrate specificity from aspartate

to alanine; however, it may not be likely. The high degree of sequence similarity between

AspAT and AlaAT with the exception of the site for mutagenesis supports the possibility that the

suggested Lys-99 residue is responsible for aspartate specificity. Docking alanine with the K99S

mutant showed a distance of 6.3 angstroms between the serine residue and alanine substrate,

comparable to that of the wild type AlaAT with a distance of 7.8 angstroms. This similarity

suggests that the same type of interaction is possible with the serine mutation in AspAT.

However, the rotamer necessary to achieve the correct orientation towards alanine only has a

12.5% likelihood of occurring. Even if a successful mutation is obtained, it is likely that the

mutation may be oriented in such a way to prevent alanine specificity. Additionally, the

Table 2. DNA Quantitation by NanoDropAbsorbanc

eConcentration A260/280 A260/230

0.796 64.2 ng/μL 1.83 1.61

argument built on sequence similarity fails to explain how a larger binding pocket will increase

specificity for a smaller substrate. In a similar study of the aspartate aminotransferase in T.

thermophilus, a K101S mutation in a series of four site-directed mutations proved a step towards

dual substrate specificity rather than removing aspartate specificity12. The K99S mutant may

show increased specificity towards alanine and decreased specificity towards aspartate; however,

it is unlikely to achieve a full alteration of substrate specificity.

The results of nanodrop quantitation and sequencing indicate a highly successful

mutation. An A260/280 of 1.83 shows no significant contamination by RNA; however, an A260/230 of

1.61 indicates some aromatic organic contaminants. These contaminants might have presented a

problem in sequencing, but sequencing results returned without issue. pBLAST of the mutant

against the wild type showed a difference of lysine to serine only at the site of mutation, leaving

all other sites conserved.

Future Directions

With a successful mutation, the next step is to grow the K99S mutant protein and

compare the substrate specificity of the mutant for both aspartate and alanine to the specificity of

the wild type aminotransferases for Thermotoga maritima. Substrate specificity can be evaluated

using a similar method to that of labs three and four conducted earlier in the Fall semester of

Chem4411 to calculate the kinetic parameters of lactate dehydrogenase. Studies have shown the

most success for aspartate aminotransferase assays employing malate dehydrogenase and NADH

for enzyme coupling.3 Observations occur at 340 nm where NADH has an absorption maximum

and NAD+ has an absorption minimum.13 Four separate studies are needed to study a change in

substrate specificity: the kinetics of the K99S with both Ala and Asp as well as the kinetics of the

wild type AspAT and AlaAT with their respective substrates for controls. The optimal

aminotransferase concentration for each enzyme is needed to determine the kinetic parameters of

the enzyme. The best enzyme concentration gives the most linear absorbance change with the

highest R2 value using a linear regression with varying concentrations of NADH. Using this

aminotransferase concentration, substrate concentration can be varied while enzyme

concentration is maintained to calculate kinetic parameters. Background rates will be measured

with the assay in the absence of aminotransferase. Specificity is defined as the ratio of kcat/km

values calculated from the fit using either the Michaelis-Menten or Hill equation.13,14

References

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Name: Julia DiOrioPartner Names: Thammana Nishith and Michael ZhangPOI: AspAT TM1255TA: Meng ZhuangDate the report is turned in: December 7, 2015

I pledge, on my honor as a student, I have neither given nor received aid on this assignment.