Part:BBa_K5089009

ABO2449(H243R) is an α/β hydrolase mutant with a H243R mutation to improve enzyme stability at acidic pH. Our team designed these mutants for application in acidic compost environments but can be used by other iGEM teams that require an esterase.

We designed these mutants inspired by Chen et al. (2019) who explored amino acid substation to arginine residues within the active site of Bacillus acidopullulyticus pullulanase to shift its pH optimum to acidic pH (from pH 5.0 – 4.0). By analyzing the hydrogen bonding network involving the catalytic residues within the active site, they identified a set of residues that formed hydrogen bonds with residues that directly interact with the catalytic residue (1).

The rationale for this approach is that arginine’s side chain can stabilize the deprotonated catalytic serine which in turn lowers the overall pKa of the enzyme towards acidic pH (1). Using this approach, we identified potential amino acid targets, including the H243R mutation in ABO2449 (Figure 1).

Figure 1. 3D representation of the ABO2449 Alphafold 3.0 generated structure. Catalytic triad residues are shown in green (S120, D247, H275). A) wildtype ABO2449 B) mutant ABO2449(H243R)

This mutant was designed to aid in improving enzyme activity under acidic pH conditions, based on the rationale explained in (1). We wanted to ensure that binding affinity was not compromised by this mutation and thus modelled interactions with our para-nitrophenyl butyrate (pNPB) substrate (Figure 2). We obtained binding affinities from AutoDock Vina and confirmed similar substrate affinity in the designed mutant as compared to wildtype (Figure 3).

Figure 2. 2D representation of the protein-ligand binding of ABO2449 [H243R] with para-nitrophenyl butyrate (pNPB).

Figure 3. Binding affinity (from AutoDock Vina) of wild-type ABO2449 docked with pNPB, compared to H243R and S146R mutants.

References: 1. Chen, A., Xu, T., Ge, Y., Wang, L., Tang, W., & Li, S. 2019. Hydrogen-bond-based protein engineering for the acidic adaptation of Bacillus acidopullulyticus pullulanase. Enzyme and Microbial Technology, 124, 79–83. https://doi.org/10.1016/j.enzmictec.2019.01.010

ABO2449(H243R)

Assembly Compatibility:

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

  Illegal BamHI site found at 735
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 754
  Illegal NgoMIV site found at 900
  Illegal AgeI site found at 258
  
• 1000
  COMPATIBLE WITH RFC[1000]