Part:BBa_K4347007:Design

Bst with point mutations for enhanced thermal stability codon optimized for E.coli

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 766
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Sequence alignment

Since Bst is structurally homologous to Klentaq polymerase, we fetched the Bst (6MU6) and Klentaq (6QV4) FASTA sequences from the Protien Data Bank and ran a multiple sequence alignment using Seaview. A good amount of conservation between the amino acid sequences was found between the two polymerases.

Protien sequence alignment of Bst (6MU5) and Klentaq (6QV4) in Seaview.

Residues to mutate

Since there is little literature about point mutations on Bst, point mutations made in Taq (Klentaq) were sought after. Three notable point mutations were found in the Klentaq thumb domain at positions K505A, K540A and K542A where each Lysine (K) residue was switched to an Arginine residue (R)^[1]. After superimposing and aligning the structures and sequences in Pymol, the corresponding positions in Bst were found:

• K505 in Klentaq = K549 in Bst
• K540 in Klentaq = K582 in Bst
• K542 in Klentaq = Q584 in Bst

To confirm if these residues in Bst were suitable to mutate, the amino acid stability was estimated using a protien simulation software YASARA. A position scan was ran for each residue to measure the change in free energy (Kcal/mol) if the other 19 amino acid residues were mutated into that position. A mutation is classified as stabilizing if the change in free energy is ≤-1 kcal/mol, it is classified as destabilizing if the change is ≥1 kcal/mol, and neutral if it falls between these values ^[2]. The changes in free energy for mutations at residues K549, K582 and K584 are shown below:

YASARA position scan results for possible point mutations at residue K549.

YASARA position scan results for possible point mutations at residue K582.

YASARA position scan results for possible point mutations at residue Q584.

Further research was conducted to narrow down on the most optimal amino acid mutation for each position. It was found that hydrophobic interactions play a role in protien thermal stability^[3], thus substitutions of amino acids with hydrophobic side chains were made in accordance to the data obtained from the YASARA simulations:

• K549 --> W549 (-0.126 kcal/mol)
• K582 --> L582 (-1.632 kcal/mol)
• Q584 --> L584 (-2.039 kcal/mol)

Design Considerations

Since the goal of these point mutations was to increase thermal stability and not necessarily improve polymerase function, residues in the polymerase active site and fingers domain were avoided. In an article by ___, it was found that only 25% of the mutations made in the fingers domain of Taq resulted in a functional polymerase whereas 70% and 60% were functional in the palm and thumb domains. Due to the sequence similarity of Bst and Taq, these inactive mutations would likely have the same effect on Bst. It was further confimred that residues

Considering which residues were in active site or not. Fingers are important for strand displacement and activity in taq(victor to fetch paper) and when mutated only 25% worked compared to thumb and palm domains.

Source

PDB: 6MU5: https://www.rcsb.org/structure/6mu5

References

1. https://patents.google.com/patent/WO2009155464A2/en

2. https://www.frontiersin.org/articles/10.3389/fbioe.2020.558247/full

3. https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05954.x