A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 29-OH of GTP.

Pratik, Rajendra Patil and Neha, Vithani and Virender, Singh and Ashok, Kumar and Balaji, Prakash (2020) A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 29-OH of GTP. Journal of Biological Chemistry, 295 (37). pp. 12851-12867.

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Abstract

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 39-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg21 ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 29-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substratebound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogs substituted with different chemical moieties at the 29 position suggest a clear role for 29-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 29-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.

Item Type:	Article
Uncontrolled Keywords:	Bacterial Rel proteins, Gpp synthesis, bacterial pathogenesis
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 16 Protein Biochemistry
Divisions:	Molecular Nutrition
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	27 Nov 2020 10:50
Last Modified:	27 Nov 2020 10:50
URI:	http://ir.cftri.res.in/id/eprint/14667

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