The structure/function relationship of a dual-substrate (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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Two-step ligand binding in a (βα)<inf>8</inf> barrel enzyme: Substrate-bound structures shed new light on the catalytic cycle of HisA
2015, Journal of Biological ChemistryCitation Excerpt :The same logic is also likely to explain why mutation of the equivalent residue in MtPriA (D130A) leads to a 20-fold decrease in kcat while not affecting Km (10). The functional importance of His-47 and Thr-171 has previously been demonstrated in HisA and/or PriA (7, 9, 10); here we have demonstrated that Ser-202 also has an important role. Ser-202 makes hydrogen bond interactions with the 3′ hydroxyl of the reacting ribose in both the open and closed structure (Figs. 3, 4, and 5 (B and C)).
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Protein Design through Systematic Catalytic Loop Exchange in the (β/α)<inf>8</inf> Fold
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