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

Shi R, McDonald L, Cui Q, Matte A, Cygler M, Ekiel I
Proc Natl Acad Sci U S A 108 1302-7 (2011)
Related entries: 3pnk, 3pnl, 3pno, 3pnq

Cited: 13 times
EuropePMC logo PMID: 21209328

Abstract

The 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 His218(K)* 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 His56(K) is involved in formation of the covalent hemiaminal bond with Dha. The structure of H56N(K) 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 His218(K) orients the substrate optimally for phosphoryl transfer. Asp109(K) is critical for activity, likely acting as a general base activating the γ-OH of Dha. Our results provide a comprehensive picture of the roles of the highly conserved active site residues of dihydroxyacetone kinases.

Articles - 3pnm mentioned but not cited (1)

  1. Structural and mechanistic insight into covalent substrate binding by Escherichia coli dihydroxyacetone kinase. Shi R, McDonald L, Cui Q, Matte A, Cygler M, Ekiel I. Proc. Natl. Acad. Sci. U.S.A. 108 1302-1307 (2011)


Reviews citing this publication (2)

  1. Regulation of histone gene expression in budding yeast. Eriksson PR, Ganguli D, Nagarajavel V, Clark DJ. Genetics 191 7-20 (2012)
  2. Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective. Lammers M. Front Microbiol 12 757179 (2021)

Articles citing this publication (10)

  1. Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus. Parsons JB, Broussard TC, Bose JL, Rosch JW, Jackson P, Subramanian C, Rock CO. Proc. Natl. Acad. Sci. U.S.A. 111 10532-10537 (2014)
  2. Canadian macromolecular crystallography facility: a suite of fully automated beamlines. Grochulski P, Fodje M, Labiuk S, Gorin J, Janzen K, Berg R. J. Struct. Funct. Genomics 13 49-55 (2012)
  3. Coiled-coil helix rotation selects repressing or activating state of transcriptional regulator DhaR. Shi R, McDonald L, Cygler M, Ekiel I. Structure 22 478-487 (2014)
  4. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family. Broussard TC, Miller DJ, Jackson P, Nourse A, White SW, Rock CO. J. Biol. Chem. 291 6292-6303 (2016)
  5. Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements. Rodrigues JR, Couto A, Cabezas A, Pinto RM, Ribeiro JM, Canales J, Costas MJ, Cameselle JC. J. Biol. Chem. 289 10620-10636 (2014)
  6. Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Escherichia coli Mutants through Expression of a Gene Variant (fsaAA129S) for Fructose 6-Phosphate Aldolase. Guitart Font E, Sprenger GA. Int J Mol Sci 21 E9625 (2020)
  7. Structural insights into the putative bacterial acetylcholinesterase ChoE and its substrate inhibition mechanism. Pham VD, To TA, Gagné-Thivierge C, Couture M, Lagüe P, Yao D, Picard MÈ, Lortie LA, Attéré SA, Zhu X, Levesque RC, Charette SJ, Shi R. J Biol Chem 295 8708-8724 (2020)
  8. A computational study of the phosphoryl transfer reaction between ATP and Dha in aqueous solution. Bordes I, Ruiz-Pernía JJ, Castillo R, Moliner V. Org. Biomol. Chem. 13 10179-10190 (2015)
  9. Closure of the Human TKFC Active Site: Comparison of the Apoenzyme and the Complexes Formed with Either Triokinase or FMN Cyclase Substrates. Rodrigues JR, Cameselle JC, Cabezas A, Ribeiro JM. Int J Mol Sci 20 (2019)
  10. Domain architecture and catalysis of the Staphylococcus aureus fatty acid kinase. Subramanian C, Cuypers MG, Radka CD, White SW, Rock CO. J Biol Chem 298 101993 (2022)


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