Substrate specificity and mechanism from the structure of Pyrococcus furiosus galactokinase

Andrew Hartley, Steven E. Glynn, Vladimir Barynin, Patrick J. Baker, Svetlana E. Sedelnikova, Corné Verhees, Daniel De Geus, John Van Der Oost, David J. Timson, Richard J. Reece, David W. Rice

Research output: Contribution to journalArticlepeer-review

Abstract

Galactokinase (GalK) catalyses the first step of the Leloir pathway of galactose metabolism, the ATP-dependent phosphorylation of galactose to galactose-1-phosphate. In man, defects in galactose metabolism can result in disorders with severe clinical consequences, and deficiencies in galactokinase have been linked with the development of cataracts within the first few months of life. The crystal structure of GalK from Pyrococcus furiosus in complex with MgADP and galactose has been determined to 2.9Å resolution to provide insights into the substrate specificity and catalytic mechanism of the enzyme. The structure consists of two domains with the active site in a cleft at the domain interface. Inspection of the substrate binding pocket identifies the amino acid residues involved in galactose and nucleotide binding and points to both structural and mechanistic similarities with other enzymes of the GHMP kinase superfamily to which GalK belongs. Comparison of the sequence of the Gal3p inducer protein, which is related to GalK and which forms part of the transcriptional activation of the GAL gene cluster in the yeast Saccharomyces cerevisiae, has led to an understanding of the molecular basis of galactose and nucleotide recognition. Finally, the structure has enabled us to further our understanding on the functional consequences of mutations in human GalK which cause galactosemia.

Original languageEnglish
Pages (from-to)387-398
Number of pages12
JournalJournal of Molecular Biology
Volume337
Issue number2
DOIs
Publication statusPublished - 19 Mar 2004

Keywords

  • F, structure factor amplitudes for the anomalous scatterers
  • Galactokinase deficiency
  • GalK, galactokinase
  • GHMP kinase superfamily
  • Pyrococcus furiosus
  • X-ray crystallography

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