Cargando…
The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’
The 1.72 Å resolution structure of purine nucleoside phosphorylase from Geobacillus stearothermophilus, a thermostable protein of potential interest for the biocatalytic synthesis of antiviral nucleoside compounds, is reported. The structure of the N-terminally His-tagged enzyme is a hexamer, as is...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
International Union of Crystallography
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9716568/ https://www.ncbi.nlm.nih.gov/pubmed/36458621 http://dx.doi.org/10.1107/S2053230X22011025 |
_version_ | 1784842719514853376 |
---|---|
author | Given, Fiona M. Moran, Fuchsia Johns, Ashleigh S. Titterington, James A. Allison, Timothy M. Crittenden, Deborah L. Johnston, Jodie M. |
author_facet | Given, Fiona M. Moran, Fuchsia Johns, Ashleigh S. Titterington, James A. Allison, Timothy M. Crittenden, Deborah L. Johnston, Jodie M. |
author_sort | Given, Fiona M. |
collection | PubMed |
description | The 1.72 Å resolution structure of purine nucleoside phosphorylase from Geobacillus stearothermophilus, a thermostable protein of potential interest for the biocatalytic synthesis of antiviral nucleoside compounds, is reported. The structure of the N-terminally His-tagged enzyme is a hexamer, as is typical of bacterial homologues, with a trimer-of-dimers arrangement. Unexpectedly, several residues of the recombinant tobacco etch virus protease (rTEV) cleavage site from the N-terminal tag are located in the active site of the neighbouring subunit in the dimer. Key to this interaction is a tyrosine residue, which sits where the nucleoside ring of the substrate would normally be located. Tag binding appears to be driven by a combination of enthalpic, entropic and proximity effects, which convey a particularly high affinity in the crystallized form. Attempts to cleave the tag in solution yielded only a small fraction of untagged protein, suggesting that the enzyme predominantly exists in the tag-bound form in solution, preventing rTEV from accessing the cleavage site. However, the tagged protein retained some activity in solution, suggesting that the tag does not completely block the active site, but may act as a competitive inhibitor. This serves as a warning that it is prudent to establish how affinity tags may affect protein structure and function, especially for industrial biocatalytic applications that rely on the efficiency and convenience of one-pot purifications and in cases where tag removal is difficult. |
format | Online Article Text |
id | pubmed-9716568 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | International Union of Crystallography |
record_format | MEDLINE/PubMed |
spelling | pubmed-97165682022-12-12 The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’ Given, Fiona M. Moran, Fuchsia Johns, Ashleigh S. Titterington, James A. Allison, Timothy M. Crittenden, Deborah L. Johnston, Jodie M. Acta Crystallogr F Struct Biol Commun Research Communications The 1.72 Å resolution structure of purine nucleoside phosphorylase from Geobacillus stearothermophilus, a thermostable protein of potential interest for the biocatalytic synthesis of antiviral nucleoside compounds, is reported. The structure of the N-terminally His-tagged enzyme is a hexamer, as is typical of bacterial homologues, with a trimer-of-dimers arrangement. Unexpectedly, several residues of the recombinant tobacco etch virus protease (rTEV) cleavage site from the N-terminal tag are located in the active site of the neighbouring subunit in the dimer. Key to this interaction is a tyrosine residue, which sits where the nucleoside ring of the substrate would normally be located. Tag binding appears to be driven by a combination of enthalpic, entropic and proximity effects, which convey a particularly high affinity in the crystallized form. Attempts to cleave the tag in solution yielded only a small fraction of untagged protein, suggesting that the enzyme predominantly exists in the tag-bound form in solution, preventing rTEV from accessing the cleavage site. However, the tagged protein retained some activity in solution, suggesting that the tag does not completely block the active site, but may act as a competitive inhibitor. This serves as a warning that it is prudent to establish how affinity tags may affect protein structure and function, especially for industrial biocatalytic applications that rely on the efficiency and convenience of one-pot purifications and in cases where tag removal is difficult. International Union of Crystallography 2022-11-28 /pmc/articles/PMC9716568/ /pubmed/36458621 http://dx.doi.org/10.1107/S2053230X22011025 Text en © Fiona Given et al. 2022 https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. |
spellingShingle | Research Communications Given, Fiona M. Moran, Fuchsia Johns, Ashleigh S. Titterington, James A. Allison, Timothy M. Crittenden, Deborah L. Johnston, Jodie M. The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’ |
title | The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’ |
title_full | The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’ |
title_fullStr | The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’ |
title_full_unstemmed | The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’ |
title_short | The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’ |
title_sort | structure of his-tagged geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’ |
topic | Research Communications |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9716568/ https://www.ncbi.nlm.nih.gov/pubmed/36458621 http://dx.doi.org/10.1107/S2053230X22011025 |
work_keys_str_mv | AT givenfionam thestructureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT moranfuchsia thestructureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT johnsashleighs thestructureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT titteringtonjamesa thestructureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT allisontimothym thestructureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT crittendendeborahl thestructureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT johnstonjodiem thestructureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT givenfionam structureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT moranfuchsia structureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT johnsashleighs structureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT titteringtonjamesa structureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT allisontimothym structureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT crittendendeborahl structureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks AT johnstonjodiem structureofhistaggedgeobacillusstearothermophiluspurinenucleosidephosphorylaserevealsaspannerintheworks |