Nonhydrolysable analogues of (p)ppGpp and (p)ppApp alarmone nucleotides as novel molecular tools

DOI: 10.1021/acschembio.1c00398

Authors: Viktor Mojr (Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences) , Mohammad Roghanian (Umeå University) , Hedvig Tamman (Université libre de Bruxelles; WELBIO) , Duy Dinh Do Pham (Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences) , Magdalena Petrová (Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences) , Radek Pohl (Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences) , Hiraku Takada (Umeå University; Kyoto Sangyo University) , Katleen Van Nerom (Université libre de Bruxelles; WELBIO) , Hanna Ainelo (Université libre de Bruxelles; WELBIO) , Julien Caballero-Montes (Université libre de Bruxelles; WELBIO) , Steffi Jimmy (Umeå University; Deutsches Elektronen-Synchrotron DESY) , Abel Garcia-Pino (Université libre de Bruxelles) , Vasili Hauryliuk (Umeå University; University of Tartu; Lund University) , Dominik Rejman (Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Chemical Biology , VOL 3

State: Published (Approved)
Published: September 2021
Diamond Proposal Number(s): 23248

Abstract: While alarmone nucleotides guanosine-3′,5′-bisdiphosphate (ppGpp) and guanosine-5′-triphosphate-3′-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3′-azido-3′-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.

Journal Keywords: Mixtures; Nucleic acids; Peptides and proteins; Bacteria; Hydrolysis

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: PROXIMA 1, PROXIMA 2A at Soleil

Added On: 07/09/2021 09:15

Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)