The structure/function relationship of a dual-substrate (βα)8-isomerase

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Abstract

Two structures of phosphoribosyl isomerase A (PriA) from Streptomyces coelicolor, involved in both histidine and tryptophan biosynthesis, were solved at 1.8 Å resolution. A closed conformer was obtained, which represents the first complete structure of PriA, revealing hitherto unnoticed molecular interactions and the occurrence of conformational changes. Inspection of these conformers, including ligand-docking simulations, allowed identification of residues involved in substrate recognition, chemical catalysis and conformational changes. These predictions were validated by mutagenesis and functional analysis. Arg19 and Ser81 were shown to play critical roles within the carboxyl and amino phosphate-binding sites, respectively; the catalytic residues Asp11 and Asp130 are responsible for both activities; and Thr166 and Asp171, which make an unusual contact, are likely to elicit the conformational changes needed for adopting the active site architectures. This represents the first report of the structure/function relationship of this (βα)8-isomerase.

Section snippets

Materials and methods

Crystallisation, X-ray data collection, structure determination and refinement. Wild-type PriA and the mutant PriA_Ile67Met, which was seleno-methionine (Se-Met) substituted, were purified and crystallised as previously reported [4]. For production of Se-Met substituted PriA, the methionine auxotrophic Escherichia coli strain B834(DE3) and M9 minimal medium were used [5]. Protein expression was induced with IPTG to 1 mM at an OD600 of 0.6, and cell-growth was allowed to continue at 293 K for 16 h.

Structure determination of a novel conformer of PriA

The Se-Met substituted (open conformer) and wild-type (closed conformer) structures of PriA are virtually identical, both in terms of their crystallographic parameters (Table S1, Supplementary Information) and the overall model they represent (0.32 Å rmsd for 199 common Cα atoms). Ordering of different loops could in principle be related to the resolution at which each structure was refined, although both structures were elucidated at 1.8 Å resolution. The Se-Met derived model was refined against

Acknowledgments

This work was funded by CONACyT, Mexico (Project No. 50952-Q) and the Royal Society, UK (Project No. 20060612184333-3475-43146). We are grateful to Filiberto Sánchez, Paul Gaytan and Jorge Yañez for technical assistance and the European Synchrotron Radiation Facility (ESRF) for access and user support.

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