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
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 0961-8368Full text not available from this repository.
Official URL: http://dx.doi.org/10.1002/pro.331
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.|
|Official Date:||March 2010|
|Number of Pages:||9|
|Page Range:||pp. 535-543|
|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|
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