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A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2′-OH of GTP

Authors
  • Patil, Pratik Rajendra1
  • Vithani, Neha1
  • Singh, Virender1
  • Kumar, Ashok1
  • Prakash, Balaji2
  • 1 Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India
  • 2 Department of Molecular Nutrition, Council of Scientific and Industrial Research–Central Food Technological Research Institute, Mysore, India
Type
Published Article
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry and Molecular Biology
Publication Date
Jul 21, 2020
Volume
295
Issue
37
Pages
12851–12867
Identifiers
DOI: 10.1074/jbc.RA120.013636
PMID: 32719004
PMCID: PMC7489905
Source
PubMed Central
Keywords
License
Unknown

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 3′-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 Mg2+ 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 2′-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound 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 2′ position suggest a clear role for 2′-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2′-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.

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