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.

Figure 1

Schematic representation of the “electrostatic gatekeeper effect”. (A) The Blastocystis wild-type SCSβ subunit with positively charged gatekeeper residues (KK) favoring adenine and enzyme is ATP-specific. (B) Gatekeeper mutant (ED) favoring both adenine and guanine with negatively charged gatekeeper residues and showing ATP and GTP specificity. (C) Combined mutant (ED+LF) favoring adenine and guanine with negatively charged gatekeeper residues, but ATP binding hindered due to π–π stacking interactions with Phe²²⁷, which therefore resulted in an exclusive GTP-specific enzyme. The water molecules are colored turquoise, and sugar and phosphate groups are not shown because of the similarities.

Figure 2

Sequence alignment of SCSβ subunits of Blastocystis, pig, and E. coli. Gatekeeper residues are highlighted in yellow, and nucleotide binding site residues are highlighted in green. Alignment is done using Clustal W.

Figure 3

Refolded and purified wild-type and mutant SCS enzymes. SCSβ and SCSα subunits are shown in the SDS–PAGE gel.

Figure 4

CD spectra of wild-type SCS and its various mutants. The CD spectra (190–260 nm) of the wild type (red), the gatekeeper mutant (ED) (green), the nucleotide binding site mutant (LF) (violet), and the combined mutant (ED+LF) (brown) are shown.

Figure 5

Enzyme kinetics of Blastocystis SCS. Michaelis–Menten plots for kinetic measurements of Blastocystis SCS with variable concentrations of ATP and GTP. Graphs show the initial rate vs ATP and GTP concentration: (A) wild-type SCS, (B) gatekeeper mutant (ED), (C) nucleotide binding site mutant (LF), and (D) combined mutant (ED+LF). In panel C, the LF mutant has a K_m higher than the highest substrate concentration used in the experiment. Replicate values for ATP and GTP are indicated in each graph from three different assays.

Figure 6

Electrostatic surface models of the SCSβ nucleotide binding region. Electrostatic surfaces of the gatekeeper region of SCSβ are indicated with black ovals. Gatekeeper region in (A) Blastocystis wild-type SCS, (B) nucleotide binding site mutant (LF), (C) gatekeeper mutant (ED), and (D) combined mutant (ED+LF) showing the effect of the change in charge in the gatekeeper region. (E) The negatively charged gatekeeper region in pig SCS and (F) the neutral gatekeeper region in E. coli SCS are shown. Red electrostatic surfaces indicate overall negative gatekeeper residues, whereas blue electrostatic surfaces indicate positive gatekeeper residues. The electrostatic surfaces were prepared by using Modeller9 V1032 and ef-surf server and visualized in PDBj viewer.

Figure 7

Snapshots of molecular dynamic simulations (frames) for the Blastocystis SCS nucleotide binding site with ATP and GTP. Figures show ATP and GTP (red color) inside the nucleotide binding site of SCS with Leu²²⁷ (green), Lys²³⁰ (blue), and Arg⁵⁸ (sky blue) in all the systems. The nucleotide binding site mutant (LF) and the combined mutant (ED+LF) have Phe²²⁷ (green) in place of Leu²²⁷.