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

Abstract

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-phospho-nononic acid (KDN-9-P) phosphatase in the biosynthetic pathway of the 9-carbon α-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 Å 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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.