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Millisecond dynamics in the allosteric enzyme imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima

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Abstract

IGPS is a 51 kDa heterodimeric enzyme comprised of two proteins, HisH and HisF, that catalyze the hydrolysis of glutamine to produce NH3 in the HisH active site and the cyclization of ammonia with N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in HisF to produce imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR). Binding of PRFAR and IGP stimulates glutaminase activity in the HisH enzyme over 5,000 and 100-fold, respectively, despite the active sites being >25 Å apart. The details of this long-range protein communication process were investigated by solution NMR spectroscopy and CPMG relaxation dispersion experiments. Formation of the heterodimer enzyme results in a reduction in millisecond motions in HisF that extend throughout the protein. Binding of lGP results in an increase in protein-wide millisecond dynamics evidenced as severe NMR line broadening and elevated R ex values. Together, these data demonstrate a grouping of flexible residues that link the HisF active site with the protein interface to which HisH binds and provide a model for the path of communication between the IGPS active sites.

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Abbreviations

AICAR:

5-Aminoimidazole-4-carboxamide ribotide

CPMG:

Carr-Purcell-Meiboom-Gill

HisF-IGPS:

Isotopically labeled HisF in complex with HisH

IGP:

Imidazole glycerol phosphate

IGPS:

Imidiazole glycerol phosphate synthase (a heterodimer of HisF and HisH)

PRFAR:

N′-[(5′-Phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide

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Acknowledgments

JPL acknowledges support from the NIH (R01-GM070823), JML was supported by an NIH biophysical training grant (5T32GM008283). We thank Joanna Dunn (Yale University) for help in constructing His-tagged HisH and Simon Lipchock for delaying his birth until after completion of this manuscript. The authors thank V. Jo Davisson for helpful suggestions.

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Authors and Affiliations

  1. Department of Chemistry, Yale University, New Haven, CT, 06520, USA

    James Lipchock & J. Patrick Loria

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06520, USA

    J. Patrick Loria

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  1. James Lipchock
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Correspondence to J. Patrick Loria.

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Lipchock, J., Loria, J.P. Millisecond dynamics in the allosteric enzyme imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima . J Biomol NMR 45, 73–84 (2009). https://doi.org/10.1007/s10858-009-9337-8

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  • DOI: https://doi.org/10.1007/s10858-009-9337-8

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