Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools
(2021) In ACS Chemical Biology 16(9). p.1680-1691- 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... (More)
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.
(Less)
- author
- organization
- publishing date
- 2021-09-03
- type
- Contribution to journal
- publication status
- published
- subject
- in
- ACS Chemical Biology
- volume
- 16
- issue
- 9
- pages
- 1680 - 1691
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- scopus:85115092887
- pmid:34477366
- ISSN
- 1554-8937
- DOI
- 10.1021/acschembio.1c00398
- language
- English
- LU publication?
- yes
- id
- 91e49eeb-b4de-4789-9bb5-48dd8d3128a9
- alternative location
- https://pubs.acs.org/doi/10.1021/acschembio.1c00398
- date added to LUP
- 2021-09-04 07:54:25
- date last changed
- 2024-04-06 08:13:04
@article{91e49eeb-b4de-4789-9bb5-48dd8d3128a9, abstract = {{<p>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.</p>}}, author = {{Mojr, Viktor and Roghanian, Mohammad and Tamman, Hedvig and Do Pham, Duy Dinh and Petrová, Magdalena and Pohl, Radek and Takada, Hiraku and Van Nerom, Katleen and Ainelo, Hanna and Caballero-Montes, Julien and Jimmy, Steffi and Garcia-Pino, Abel and Hauryliuk, Vasili and Rejman, Dominik}}, issn = {{1554-8937}}, language = {{eng}}, month = {{09}}, number = {{9}}, pages = {{1680--1691}}, publisher = {{The American Chemical Society (ACS)}}, series = {{ACS Chemical Biology}}, title = {{Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools}}, url = {{http://dx.doi.org/10.1021/acschembio.1c00398}}, doi = {{10.1021/acschembio.1c00398}}, volume = {{16}}, year = {{2021}}, }