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Identification and analysis of residues contained on β → a loops of the dual-substrate (βα)8 phosphoriblosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity
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Noda-Garcia, Lianet, Camacho-Zarco, Aldo R., Verdel-Aranda, Karina, Wright, Helena, Soberon, Xavier, Fulop, Vilmos and Barona-Gomez, Francisco. (2010) Identification and analysis of residues contained on β → a loops of the dual-substrate (βα)8 phosphoriblosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity. Protein Science, Vol.19 (No.3). pp. 535-543. ISSN 0961-8368
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Official URL: http://dx.doi.org/10.1002/pro.331
Abstract
A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (βα)8 phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis–Menten enzyme kinetics for both isomerase activities in wild-type PriA from S. coelicolor and in selected single-residue monofunctional mutants, identified after Escherichia coliin vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important β → α loop 5, namely, Arg139, which was postulated on structural grounds to be important for the dual-substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser81Thr and PriA_Arg139Asn showed that these residues, which are contained on β → α loops and in close proximity to the N-terminal phosphate-binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X-ray crystallographic structure of PriA_Arg139Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this β → α loop as a major structural feature related to the evolution of the dual-substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates—within a bifunctional and thus highly constrained active site—without compromising its structural stability.
| Item Type: | Journal Article |
|---|---|
| Subjects: | Q Science > QD Chemistry |
| Divisions: | Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010) |
| Journal or Publication Title: | Protein Science |
| Publisher: | John Wiley & Sons Ltd. |
| ISSN: | 0961-8368 |
| Date: | March 2010 |
| Volume: | Vol.19 |
| Number: | No.3 |
| Number of Pages: | 9 |
| Page Range: | pp. 535-543 |
| Identification Number: | 10.1002/pro.331 |
| Status: | Peer Reviewed |
| Publication Status: | Published |
| Access rights to Published version: | Restricted or Subscription Access |
| Funder: | Royal Society (Great Britain), Conacyt, Mexico, Royal Society, United Kingdom |
| Grant number: | 50952-0, 83039 |
| URI: | http://wrap.warwick.ac.uk/id/eprint/6290 |
Data sourced from Thomson Reuters' Web of Knowledge
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