Identification and analysis of residues contained on β → α loops of the dual-substrate (βα)8 phosphoribosyl isomerase a specific for its phosphoribosyl anthranilate isomerase activity

Lianet Noda-García, Aldo R. Camacho-Zarco, Karina Verdel-Aranda, Helena Wright, Xavier Soberón, Vilmos Fülöp*, Francisco Barona-Gómez

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

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 coli in 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. Published by Wiley-Blackwell.

Original languageAmerican English
Pages (from-to)535-543
Number of pages9
JournalProtein Science
Volume19
Issue number3
DOIs
StatePublished - Mar 2010
Externally publishedYes

Keywords

  • (βα)-barrels
  • Conformational diversity
  • Dual-substrate specificity
  • HisA and TrpF
  • Loops motion
  • Phosphoribosyl isomerase A (PriA)

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