Aeropyrum pernix d-2-deoxyribose-5-phosphate aldolase (DERA) can be subjected to mutagenesis to improve its utility as a biocatalyst for drug manufacturing. [Wada 2003, Jennewein 2006, DeSantis 2003, DeSantis 2002]. Decreasing the amount of enzyme needed (and thus the amount of culture which produces the enzyme) is the major goal of mutagenesis. This can be accomplished both by increasing the stability of the enzyme (a single enzyme will be able to "turn over" more molecules of substrate before it denatures), and by increasing the binding affinity for the enzyme to its substrate. Additionally, mutagenesis to decrease the enzyme specificity will improve its ability to accept the appropriate substrate(s) for the synthesis of statin precursors [DeSantis 2002]. Mutations in A. pernix DERA will be selectively introduced at residues which are important in the enzyme active site or related to protein stability and selectivity as identified by the A. pernix DERA crystal structure and its comparison to E.coli DERA [Sakuraba 2007, Sakuraba 2003]. Once each mutant is evaluated based on the previous criteria, successful mutations will be combined into a single gene and evaluated again to confirm that the effects of the mutations are additive. These efforts will make possible the use of this enzyme to synthesize specific statin precursors and will improve the overall efficiency synthesis. This will reduce the overall consumption of the enzyme, reducing the cost, resources, and therefore the energy required to carry out the synthesis of the Atorvastatin precursor.
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A.pernix DERA will be subjected to mutagenesis targeted toward active site, and substrate-binding , site residues gene in order to expand the substrate selectivity and catalytic efficiency of the enzyme [Wada 2003, Jennewein 2006, DeSantis 2003, DeSantis 2002] for use in yeast as an industrial biocatalyst for the synthesis of a precursor of Atorvastatin.
For site-directed mutagenesis, several specific residues and groups of residues will be targeted. These have been chosen based on knowledge of the enzyme crystal structure and comparison of the A. pernix DERA structure to that of the homologous protein from E. coli, for which there is more structural data [Sakuraba 2007, Sakuraba 2003, Heine 2004]. In both proteins, a phosphate-binding pocket has been identified which is partially responsible for the specificity of DERA for its native substrate D-glyceraldehyde 3-phosphate. Without this substrate specificity, E. coli DERA has been shown to accept several different aldehydes in addition to the native substrate [DeSantis 2002]. Phosphate-binding residues in A. pernix DERA will be mutated to remove substrate specificity.
A.pernix DERA was chosen as a starting point for mutagenesis because of several key differences between this enzyme and E. coli DERA. A. pernix DERA is both far more stable and retains much more of its catalytic activity at higher temperatures than E. coli DERA [Sakuraba 2007]. Several structural differences between E. coli and A. pernix DERA have been suggested to be responsible for the difference in enzyme stability. Specifically, hydrophobic interactions in the dimer interface region are thought to help stabilize the protein [Sakuraba 2007]. Residues in this region will be mutated to several different hydrophobic residues in an attempt to improve the strength of these interactions and increase stability. To improve enzyme catalytic activity, several modifications will be made to the active site. Residues that make up a hydrophobic pocket near the backbone of the Schiff base Lys167 in the catalytic active site will be mutated to increase the hydrophobic character of the pocket. It has been observed that an increase in the hydrophobicity of this pocket may account for increased catalytic activity of a mutant E. coli DERA using chloroacetaldehyde, a starting material for Atorovastatin synthesis, as a substrate [Jennewein 2006]. Further modification of the pocket may lead to increased activity of A. pernix DERA using this non-native substrate. Additionally, residues adjacent to a key Schiff base Lysine (Lys167) in the active site will be mutated to adjust its pKa.
Site directed mutagenesis will be carried out using PCR, the PCR product cloned and transformed into E.coli, and the gene product isolated for analysis.
Mutants will be screened for temperature stability and stability in the presence of high levels of chloroacetaldehyde, using circular dichroism spectroscopy to monitor denaturation. The binding affinity of the enzyme to chloroacetaldehyde will also be a screening criterion and will be tested using isothermal titration calorimetry (ITC). Mutations which attain either of our goals will be counted as "hits" and recombined into a single gene, again using PCR. This gene product will then be re-evaluated to ensure the effects of the beneficial mutations are additive and do not interfere with one another.
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A. pernix d-2-deoxyribose-5-phosphate aldolase will be subjected to site-directed [Zheng 2004, Chen 2001] mutagenesis procedures using PCR to improve its utility as an industrial biocatalyst, i.e. improve its stability, catalytic efficiency, and expand its substrate specificity to include substrates for stain synthesis. Structural studies of A. pernix DERA have identified areas in the protein which are crucial for each of these functions. The crystal structure of A. pernix DERA [Sakuraba 2007, Sakuraba 2003] has identified several key residues in the active site, both in the catalytic portion and the substrate-binding portion, and regions in the dimer interface that will be targets for mutagenesis. Specifically, Lys 201, which affects the pKa of the Shiff base-forming Lys167 in the active site [Sakuraba 2007] will be mutated to other positively charged residues, arginine and histidine, in an attempt to tune the pKa of Lys 167 to optimize catalysis. Residues which make up a small hydrophobic pocket near the backbone of Lys 167 have been shown to affect E.coli DERA activity using chloroacetaldehyde as a substrate when mutated to more hydrophobic residues such as isoleucine [Jennewein 2006]. In A. pernix DERA, Val200, Ile166, and Ile185 make up this pocket. Val200 will be mutated to isoleucine to maximize the hyrophobicity without drastically altering side chain bulkiness. Ser238, which makes a direct hydrogen bond contact to the phosphate group of the natural substrate in the substrate-binding region [Jennewein 2006] will be mutated to glycine to remove the hydrogen bonding ability of its side chain. This mutant should show reduced specificity for the natural phosphate-containing substrate. Residues involved in hydrophobic interactions at the dimer interface, Phe59, Pro60, Phe61, and Phe151 are thought to be partially responsible for the increased thermal stability of A. pernix DERA [Sakuraba 2007]. Each of these residues will be mutated to residues with very high hydrophobicities, namely isoleucine, tryptophan, and phenylalanine in the case of Pro60 to improve the hydrophobic interactions between subunits and increase protein stability. Site-directed mutagenesis will focus on these residues with the goals of expanding substrate binding affinity and stability [DeSantis 2003].
Mutants will be overexpressed in E.coli and screened for increased activity using chloroacetaldehyde and acetaldehyde as substrates instead of the natural substrate, 2-deoxy-D-ribose 5-phosphate, as well as for increased thermal stability, and stability in the presence of high concentrations of chloroacetaldehyde. Isothermal titration calorimetry (ITC) will be used to measure the affinity of the DERA mutants for chloroacetaldehyde. Circular dichroism (CD) will be used to assay stability. "Hit" mutations will be recombined using site-directed mutagenesis [Jennewein 2006] into a single gene for use in transfection into yeast.
This mutagenesis procedure should produce a variety of DERA variants which are mutated in different ways. One would expect the variants whose mutations are in involved in the catalytic site to have some altered cayalytic activity, the Gly238 mutant to have reduced specificity to phosphate-containing substrates, and the variants with mutations in the dimer interface to have different properties of stability [Sakuraba 2007]. However, one would not expect all of the mutations to necessarily improve these qualities. Most likely only a few mutations will result in the desired outcome and the others will either be ineffective or negatively impact some desirable property of the protein. Assuming that many mutations are not incorporated into the final recombined variant at once, we expect the benefits of each individual mutation to be additive [Jennewein 2006].
Most of the potential pitfalls of the proposed experimental design involve the possible failure of mutagenesis to produce a variant which improves upon the existing A. pernix DERA. Although the mutations are targeted to specific regions of the protein with known structure and function, it may be that none of the mutants will improve the stability or catalytic activity of the protein. The mutations may certainly change the function of the enzyme, but whether or not that change will be a positive one for our purposes is uncertain. There is also a risk that when recombined, mutations that may have been beneficial on their own will interfere with one another when recombined in a single gene. This risk is especially high if the mutations for the different variants are in the same region of the protein or if they both involve residues which closely interact. If they are both in the dimer interface region, or both involved in the catalytic site, for example, the mutations could cancel out to some degree. If this occurs, it may not be possible to recombine these mutant genes into a single product which exhibits the benefits of both mutations.
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Site-directed mutants will be produced using a PCR method where specific residues are targeted and mutations introduced using tailored PCR primers [DeSantis 2003, Zheng 2004]. Primers are designed based on the sequence of the A. pernix DERA gene and can cause nucleic acid substitutions at specific sites. When the PCR cycle is run, the primers will cause the A. pernix DERA gene to be copied with the substitution introduced. This substitution is then amplified in subsequent PCR cycles. The PCR products will be purified by electrophoresis.
The following residues are identitified by the A. pernix crystal structure [Sakuraba 2003, Sakuraba 2007] and will be targeted for site-directed mutagenesis: Lys201 will be mutated to His201 and Arg201, Ser238 will be mutated to Gly238, and Val200 will be mutated to Ile200. Phe59, Pro60, Phe61, and Phe151 will be mutated in separate variants to Trp or Ile59, Phe60, Trp or Ile61, and Trp or Ile151, respectively. Together, there will be eleven mutants.
The PCR product for each mutation will be overexpressed in E.coli and the gene product purified for analysis of stability and binding affinity.
Each mutant will be tested for thermal stability, stability in the presence of high concentrations of chloroacetaldehyde, and binding affinity for chloroacetaldehyde. Stability measurements will be determined using circular dichroism (CD) spectroscopy. Elipticity measurements of solutions of each mutant protein as well as the wild type protein will be taken from 190 nm to 250nm at room temperature to determine a wavelength where the spectrum differs from that of a random coil. Because A. pernix DERA contains mostly alpha helices and beta sheets [Sakuraba 2003, DeSantis 2002], we can expect the ellipticity at 195 nm to be positive and this will most likely be our wavelength of choice for each mutant. The ellipticity at this wavelength will then be monitored after the sample has been elevated to 100Â°C, and every 10 minutes for several hours while the high temperature is sustained. Because A.pernix is hyperthermophilic, we expect the thermal stability to already be fairly high, hence the sustained high temperature. A decrease in ellipticity at our wavelength of choice will indicate that the protein is converting to a random coil configuration, and therefore the protein has denatured. The rate of denaturation for each mutant will be compared to the wild type protein to determine if the mutation in question improved or worsened thermal stability. A similar procedure will be employed to test for stability in the presence of high concentrations of chloroacetaldehyde except instead of elevating the temperature, 300mM chloroacetaldehyde will included in the protein solution and the ellipticity will be monitored over a longer period of time, perhaps as long as 20 hours [Sakuraba 2007]. Similarly, the rate of denaturation in the presence of chloroacetaldehyde will be compared to the wild-type protein to determine if the mutation in question improved stability.
Affinity for chloroacetaldehyde will be tested using isothermal titration calorimetry. Small quantities of chloroacetaldehyde will be injected into an ITC sample cell containing the each mutant protein as well as the wild type protein. A binding isotherm will be produced, from which the Kd can be extracted. The Kds of the mutants will be compared to that of the wild type. For our purposes, "hits" will be mutants that exhibit greater binding affinity for chloroacetaldehyde (have a lower Kd) than the wild type. These mutants will have a reduced specificity for phosphate-containing substrates and will bind synthetically relevant acetaldehydes more readily.
"Hit" mutant genes will be recombined by using the same site-directed PCR technique to make multiple "hit" mutations in the same gene [Zheng 2004, DeSantis 2003]. The gene will be amplified by PCR, expressed in E. coli, and purified as above. If more than one different mutation of the same residue (for example, the His201 and Arg 201 mutants) are "hits", then more than one recombinant DERA will be made. The recombinants protein(s) will be subjected to the same screening tests described above to characterize the interaction of their multiple mutations. The final product of mutagenesis will be a protein which is both more stable than wild-type A. pernix DERA and has a higher affinity for statin synthesis starting materials, namely chloroacetaldehyde.