Charged gatekeeper residues alone can’t determine the nucleotide
specificity of succinyl-CoA synthetase (SCS) of P. falciparum
Abstract
Understanding the molecular basis of substrate specificity of an enzyme
is very crucial to its functional importance in any biological system.
Previously, we had experimentally validated a novel phenomenon using an
enzyme engineering approach, to alter the substrate specificity via
modification of the electrostatic properties of the gatekeeper residues
in succinyl-CoA synthetase (SCS) of Blastocystis. The multiple sequence
alignment (MSA) of SCSβ subunits from phylogenetically diverse
organisms, depicted P. falciparum SCS to club with ADP-forming human,
bovine and murine SCS enzymes. In the present study, we have explored
the gatekeeper residues of P. falciparum SCS (PfSCS), an enzyme crucial
for the generation of a metabolic intermediate- succinyl-CoA, required
during the mosquito stages of the Plasmodium. With the construction of
various gatekeeper mutants, structural modeling and enzyme kinetics
experiments, our study concluded that PfSCS is an ADP-forming enzyme
(KmATP=48 µM). Introduction of the exclusively charged- positive (Lys &
Lys) & negative gatekeeper residues (Glu & Asp), demonstrated
significant reductions in the ATP affinity, while no significant
GDP-forming potential was recorded. Interestingly, simultaneous
refolding of the nucleotide-binding site containing PfSCSβ subunit with
the Blastocystis SCSα produced the active enzyme conformation.
Therefore, the present study concluded that only electrostatic
interactions at the gatekeeper region are not sufficient enough to alter
the substrate specificity in PfSCS, as in case of Blastocystis SCS and
further structural analysis is warranted with particular focus on the
binding site architecture of PfSCS.