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Salomone-Stagni, Marco, Bartho, Joseph D., Kalita, Eeshan, Rejzek, Martin, Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275, Bellini, Dom, Walsh, Martin A. and Benini, Stefano
(2018)
Structural and functional analysis of Erwinia amylovora SrlD. The first crystal structure of a sorbitol-6-phosphate 2-dehydrogenase.
Journal of Structural Biology, 203 (2).
pp. 109-119.
ISSN 1047-8477
Abstract
Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of D-sorbitol 6-phosphate to D-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Å resolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing D-sorbitol 6-phosphate than in reducing D-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the D-sorbitol 6-phosphate present in the in vitro environment is converted into D-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.
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| Additional Information: | Funding Information: The work of MSS was funded by the MESCAL project from the Free University of Bolzano – Italy (grant number: 1440 ) while the consumables costs were funded by the “A structural genomics approach for the study of the virulence and pathogenesis of Erwinia amylovora ” project from the Autonomous Province of Bolzano – Italy (grant number: 98 ). JDB was recipient of a predoctoral fellowship from the Free University of Bolzano. Work at the John Innes Centre was supported by the UK BBSRC Institute Strategic Programme on Understanding and Exploiting Metabolism (MET) [BB/J004561/1] and the John Innes Foundation . We thank the Diamond Light Source for the beam time provided. We thank Dr Luca Daprà for preliminary work on SrlD. Appendix A Funding Information: The work of MSS was funded by the MESCAL project from the Free University of Bolzano – Italy (grant number: 1440) while the consumables costs were funded by the “A structural genomics approach for the study of the virulence and pathogenesis of Erwinia amylovora” project from the Autonomous Province of Bolzano – Italy (grant number: 98). JDB was recipient of a predoctoral fellowship from the Free University of Bolzano. Work at the John Innes Centre was supported by the UK BBSRC Institute Strategic Programme on Understanding and Exploiting Metabolism (MET) [BB/J004561/1] and the John Innes Foundation. We thank the Diamond Light Source for the beam time provided. We thank Dr Luca Daprà for preliminary work on SrlD. Funding Information: The work of MSS was funded by the MESCAL project from the Free University of Bolzano ? Italy (grant number: 1440) while the consumables costs were funded by the ?A structural genomics approach for the study of the virulence and pathogenesis of Erwinia amylovora? project from the Autonomous Province of Bolzano ? Italy (grant number: 98). JDB was recipient of a predoctoral fellowship from the Free University of Bolzano. Work at the John Innes Centre was supported by the UK BBSRC Institute Strategic Programme on Understanding and Exploiting Metabolism (MET) [BB/J004561/1] and the John Innes Foundation. We thank the Diamond Light Source for the beam time provided. We thank Dr Luca Dapr? for preliminary work on SrlD. Publisher Copyright: © 2018 Elsevier Inc. |
| Uncontrolled Keywords: | d-sorbitol 6-phosphate,fire blight,kinetics,phytopathology,rosaceae,x-ray crystallography,structural biology ,/dk/atira/pure/subjectarea/asjc/1300/1315 |
| Faculty \ School: | Faculty of Science > School of Chemistry, Pharmacy and Pharmacology |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 04 Sep 2024 08:34 |
| Last Modified: | 25 Sep 2024 18:06 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96505 |
| DOI: | 10.1016/j.jsb.2018.03.010 |
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