Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools.

Autor: Mojr V; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic., Roghanian M; Laboratory for Molecular Infection Medicine Sweden (MIMS) and Umea° Centre for Microbial Research (UCMR), Umeå University, 901 87 Umeå, Sweden.; Department of Clinical Microbiology, Rigshospitalet, 2200 Copenhagen, Denmark., Tamman H; Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles, Campus La Plaine, Building BC, (1C4 203), Boulevard du Triomphe, 1050, Brussels, Belgium.; WELBIO, Avenue Hippocrate 75, 1200 Brussels, Belgium., Do Pham DD; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic., Petrová M; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic., Pohl R; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic., Takada H; Laboratory for Molecular Infection Medicine Sweden (MIMS) and Umea° Centre for Microbial Research (UCMR), Umeå University, 901 87 Umeå, Sweden.; Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo, Motoyama, Kita-ku, Kyoto 603-8555, Japan., Van Nerom K; Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles, Campus La Plaine, Building BC, (1C4 203), Boulevard du Triomphe, 1050, Brussels, Belgium.; WELBIO, Avenue Hippocrate 75, 1200 Brussels, Belgium., Ainelo H; Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles, Campus La Plaine, Building BC, (1C4 203), Boulevard du Triomphe, 1050, Brussels, Belgium.; WELBIO, Avenue Hippocrate 75, 1200 Brussels, Belgium., Caballero-Montes J; Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles, Campus La Plaine, Building BC, (1C4 203), Boulevard du Triomphe, 1050, Brussels, Belgium.; WELBIO, Avenue Hippocrate 75, 1200 Brussels, Belgium., Jimmy S; Laboratory for Molecular Infection Medicine Sweden (MIMS) and Umea° Centre for Microbial Research (UCMR), Umeå University, 901 87 Umeå, Sweden.; Deutsches Elektronen-Synchrotron DESY, Centre for Structural Systems Biology (CSSB), Notkestr. 85, 22607 Hamburg, Germany., Garcia-Pino A; Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles, Campus La Plaine, Building BC, (1C4 203), Boulevard du Triomphe, 1050, Brussels, Belgium.; WELBIO, Avenue Hippocrate 75, 1200 Brussels, Belgium., Hauryliuk V; Laboratory for Molecular Infection Medicine Sweden (MIMS) and Umea° Centre for Microbial Research (UCMR), Umeå University, 901 87 Umeå, Sweden.; University of Tartu, Institute of Technology, 50411 Tartu, Estonia.; Department of Experimental Medical Science, Lund University, 221 00 Lund, Sweden., Rejman D; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic.
Jazyk: angličtina
Zdroj: ACS chemical biology [ACS Chem Biol] 2021 Sep 17; Vol. 16 (9), pp. 1680-1691. Date of Electronic Publication: 2021 Sep 03.
DOI: 10.1021/acschembio.1c00398
Abstrakt: 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)ppNu N pp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppG N pp as a molecular tool, we show that (i) as an HD substrate mimic, ppG N pp 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)ppG N pp 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 pppG N pp, 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.
Databáze: MEDLINE
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