Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues

Abstract

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called “electrostatic gatekeeper effect”. This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the “electrostatic gatekeeper effect”. These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

ATP-specificity of succinyl-CoA synthetase fromBlastocystis hominis

“…In contrast, the presence of GTP or GDP did not stabilize the protein, giving no indication that either nucleotide bound to the K46E/K114D double mutant. The results obtained from both the crystal structures and DSF differ from the results of the kinetic assays, which showed that GTP served as a substrate (Vashisht et al, 2017). One possible explanation is that the different results were owing to the different proteins used.…”

“…There are no significant structural differences between wild-type Abd-BhSCS and the K46E, K114D and V113L mutants, but the L227F mutant differs in that no magnesium is bound. This change in the structure could account for the higher apparent K m for ATP of the V113L/L227F double mutant (265 AE 50 mM versus 145 AE 47 mM; Vashisht et al, 2017). Kinetic assays for the K46E/ K114D double mutant showed that it can use either GTP or ATP but prefers GTP (143 AE 17 mM versus 230 AE 34 mM; Vashisht et al, 2017).…”

“…This change in the structure could account for the higher apparent K m for ATP of the V113L/L227F double mutant (265 AE 50 mM versus 145 AE 47 mM; Vashisht et al, 2017). Kinetic assays for the K46E/ K114D double mutant showed that it can use either GTP or ATP but prefers GTP (143 AE 17 mM versus 230 AE 34 mM; Vashisht et al, 2017). Although the conditions for crystallization are not the same as the conditions for the kinetic assays, GDP was expected to bind to the K46E/K114D double mutant because ADP was seen to bind to this mutant.…”

“…Positively charged residues outside the catalytic site, Lys42 and Lys110, were suggested to destabilize the binding of GTP via an electrostatic gatekeeper mechanism, leading to specificity for ATP. In subsequent experiments, these gatekeeper residues were mutated to Glu and Asp, respectively, which are the residues in similar locations in pig GTPSCS (Vashisht et al, 2017). The double mutant bound either ATP or GTP.…”

The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone

“…In 2008, Hamblin et al proposed an electrostatic gatekeeper effect, in which the nucleotide access was controlled by the charged amino acids (gatekeeper residues) outside the binding site of the succinyl-CoA synthetase (SCS) of Blastocystis-a human intestinal parasite [6]. Recently, we have experimentally demonstrated the 'electrostatic gatekeeper effect', where the gatekeeper residues were found to be critical for nucleotide specificity in Blastocystis SCS [7]. Interestingly, this study also established a novel enzyme engineering approach, where the switching of the charge of the gatekeeper residues from positive to negative, demonstrated that the ADP-forming SCS could also utilize GTP.…”