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dc.contributor.authorVashisht, K
dc.contributor.authorVerma, S
dc.contributor.authorGupta, S
dc.contributor.authorLynn, AM
dc.contributor.authorDixit, R
dc.contributor.authorMishra, N
dc.contributor.authorValecha, N
dc.contributor.authorHamblin, KA
dc.contributor.authorMaytum, R
dc.contributor.authorPandey, KC
dc.contributor.authorvan der Giezen, M
dc.date.accessioned2017-04-26T07:33:26Z
dc.date.issued2017-01-24
dc.description.abstractCharged, 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.en_GB
dc.description.sponsorshipWork is supported by the National Institute of Malaria Research, Indian Council of Medical Research, New Delhi and Dept. of Biotechnology, New Delhi. K.C.P. is a recipient of the Prof. Ramalingaswami Fellowship (Department of Biotechnology, Government of India (BT/HRD/35/02/2006), K.V. is a recipient of UGC Senior Research Fellowship, M.v.d.G. is grateful for support from the University of Exeter and the Wellcome Trust (078566/A/05/Z).en_GB
dc.identifier.citationVol. 56, Iss. 3, pp. 534 - 542en_GB
dc.identifier.doi10.1021/acs.biochem.6b00098
dc.identifier.urihttp://hdl.handle.net/10871/27255
dc.language.isoenen_GB
dc.publisherAmerican Chemical Societyen_GB
dc.relation.urlhttps://www.ncbi.nlm.nih.gov/pubmed/27478903en_GB
dc.rightsCopyright © 2016 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.en_GB
dc.titleEngineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.en_GB
dc.typeArticleen_GB
dc.date.available2017-04-26T07:33:26Z
dc.identifier.issn0006-2960
exeter.place-of-publicationUnited Statesen_GB
dc.descriptionPublisheden_GB
dc.descriptionJournal Articleen_GB
dc.descriptionThis is the final version of the article. Available from American Chemical Society via the DOI in this record.en_GB
dc.identifier.eissn1520-4995
dc.identifier.journalBiochemistryen_GB


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