Structural and mechanistic insight into covalent substrate binding by Escherichia coli dihydroxyacetone kinase

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DOIResolve DOI: http://doi.org/10.1073/pnas.1012596108/-/DCSupplemental.
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TypeArticle
Journal titlePNAS
Volume108
Issue4
Pages13021307; # of pages: 6
AbstractThe Escherichia coli dihydroxyacetone (Dha) kinase is an unusual kinase because (i) it uses the phosphoenolpyruvate carbohydrate: phosphotransferase system (PTS) as the source of high-energy phosphate, (ii) the active site is formed by two subunits, and (iii) the substrate is covalently bound to His218K* of the DhaK subunit. The PTS transfers phosphate to DhaM, which in turn phosphorylates the permanently bound ADP coenzyme of DhaL. This phosphoryl group is subsequently transferred to the Dha substrate bound to DhaK. Here we report the crystal structure of the E. coli Dha kinase complex, DhaK–DhaL. The structure of the complex reveals that DhaK undergoes significant conformational changes to accommodate binding of DhaL. Combined mutagenesis and enzymatic activity studies of kinase mutants allow us to propose a catalytic mechanism for covalent Dha binding, phosphorylation, and release of the Dha-phosphate product. Our results show that His56K is involved in formation of the covalent hemiaminal bond with Dha. The structure of H56NK with noncovalently bound substrate reveals a somewhat different positioning of Dha in the binding pocket as compared to covalently bound Dha, showing that the covalent attachment to His218K orients the substrate optimally for phosphoryl transfer. Asp109K is critical for activity, likely acting as a general base activating the-OH of Dha. Our results provide a comprehensive picture of the roles of the highly conserved active site residues of dihydroxyacetone kinases.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada; NRC Biotechnology Research Institute
Peer reviewedYes
NPARC number17673429
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Record identifier401ed0fe-ca11-4687-8b5a-ac74c0c5b69e
Record created2011-04-05
Record modified2016-05-09
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