Engineering nucleotide specificity of succinyl-CoA synthetase in blastocystis: the emerging role of gatekeeper residues
Authors
Vashisht, KapilVerma, Sonia
Gupta, Sunita
Lynn, Andrew M.
Dixit, Rajnikant
Mishra, Neelima
Valecha, Neena
Hamblin, Karleigh
Maytum, Robin
Pandey, Kailash C.
van der Giezen, Mark
Affiliation
National Institute of Malaria Research, IndiaJawaharlal Nehru University
University of Exeter
University of Bedfordshire
Issue Date
2017-01-24
Metadata
Show full item recordAbstract
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.Citation
Vashisht K, Verma S, Gupta S, Lynn AM, Dixit R, Mishra N, Valecha N, Hamblin KA, Maytum R, Pandey KC, van der Giezen M. (2017) 'Engineering nucleotide specificity of succinyl-CoA synthetase in blastocystis: the emerging role of gatekeeper residues.', Biochemistry, 56 (3), pp.534-542.Publisher
American Chemical SocietyJournal
BiochemistryPubMed ID
27478903PubMed Central ID
PMC5404824Additional Links
https://pubs.acs.org/doi/10.1021/acs.biochem.6b00098Type
ArticleLanguage
enISSN
0006-2960ae974a485f413a2113503eed53cd6c53
10.1021/acs.biochem.6b00098
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