Structure-function analysis of 2-keto-3-deoxy-D-glycero-D-galactonononate-9-phosphate phosphatase defines specificity elements in type C0 haloalkanoate dehalogenase family members

J Biol Chem. 2009 Jan 9;284(2):1224-33. doi: 10.1074/jbc.M807056200. Epub 2008 Nov 5.


The phosphotransferases of the haloalkanoate dehalogenase superfamily (HADSF) act upon a wide range of metabolites in all eukaryotes and prokaryotes and thus constitute a significant force in cell function. The challenge posed for biochemical function assignment of HADSF members is the identification of the structural determinants that target a specific metabolite. The "8KDOP" subfamily of the HADSF is defined by the known structure and catalytic activity of 2-keto-3-deoxy-8-phospho-d-manno-octulosonic acid (KDO-8-P) phosphatase. Homologues of this enzyme have been uniformly annotated as KDO-8-P phosphatase. One such gene, BT1713, from the Bacteroides thetaiotaomicron genome was recently found to encode the enzyme 2-keto-3-deoxy-d-glycero-d-galacto-9-phosphonononic acid (KDN-9-P) phosphatase in the biosynthetic pathway of the 9-carbon alpha-keto acid, 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN). To find the structural elements that provide substrate-specific interactions and to allow identification of genomic sequence markers, the x-ray crystal structures of BT1713 liganded to the cofactor Mg(2+)and complexed with tungstate or VO(3)(-)/Neu5Ac were determined to 1.1, 1.85, and 1.63 A resolution, respectively. The structures define the active site to be at the subunit interface and, as confirmed by steady-state kinetics and site-directed mutagenesis, reveal Arg-64(*), Lys-67(*), and Glu-56 to be the key residues involved in sugar binding that are essential for BT1713 catalytic function. Bioinformatic analyses of the differentially conserved residues between BT1713 and KDO-8-P phosphatase homologues guided by the knowledge of the structure-based specificity determinants define Glu-56 and Lys-67(*) to be the key residues that can be used in future annotations.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteroidetes / enzymology
  • Bacteroidetes / genetics
  • Catalytic Domain
  • Crystallography, X-Ray
  • Kinetics
  • Ligands
  • Phosphoric Monoester Hydrolases / chemistry*
  • Phosphoric Monoester Hydrolases / classification
  • Phosphoric Monoester Hydrolases / genetics
  • Phosphoric Monoester Hydrolases / metabolism*
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Substrate Specificity


  • Ligands
  • Phosphoric Monoester Hydrolases

Associated data

  • PDB/3E81
  • PDB/3E84
  • PDB/3E8M