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-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.

FIGURE 1.

Schematic of the pathways for biosynthesis and charging of the sialic acid derivatives KDO-8-P in bacteria (left), Neu5Ac-9-P in eukaryotes (center), and KDN-9-P in bacteria (right).

FIGURE 2.

X-ray crystal structure of wild-type KDN-9-P phosphatase liganded to the cofactor Mg²⁺ depicted as a ribbon diagram. A, single protomer is depicted highlighting the conserved phosphoryl transfer residues in the active site loops as follows: loop 1, Asp-10 (red) and Asp-12 (silver); loop 2, Thr-54 (green); loop 3, Lys-80 (yellow); and loop 4, Asp-103 (cyan). The β2 and β3 insert domain forms the barrel domain that allows oligomerization. B, biological tetramer with each subunit differentially colored. The active site is indicated by the position of Mg²⁺ (magenta sphere). C, close-up view of the hydrophobic packing interactions in the barrel domain. All protein images were rendered using MOLSCRIPT (30) and POVRAY.

FIGURE 3.

The active site of KDN-9-P phosphatase liganded to the cofactor Mg²⁺ highlighting the position of the active site at the subunit interface of the Rossmann fold domains. The ribbon diagram with residues depicted in ball-and-stick shows the interaction of the “catalytic domain” (gray) and “cap domain” (brown with residues denoted as ^*). The active site Mg²⁺ is depicted as a magenta sphere.

FIGURE 4.

KDN-9-P phosphatase liganded complexes. The 2F_o - F_c composite omit electron density map contoured at 1.0σ (gray mesh) is shown for all ligands and Asp-10 residue in A and B. A, close up stereo view of the active site of the cofactor Mg²⁺ and product analogue tungstate. The tungstate ion (W in black) is shown as ball-and-stick and the cofactor Mg²⁺ ion as a magenta sphere. B, close up stereo view of the active site of the complex with (green V) and Neu5Ac (yellow). C, stereo view of the active site of KDN-9-P phosphatase liganded to /Neu5Ac (yellow) and consisting of a catalytic domain (gray) from one subunit and a cap domain (brown and residues labeled as ^*) from the adjacent subunit.

FIGURE 5.

Overlay of the active site of KDN-9-P phosphatase (catalytic domain, gray; cap domain, dark gray) liganded to VO ^-₃/Neu5Ac (yellow backbone) with that of KDO-8-P phosphatase (catalytic domain, cyan; cap domain, blue). The C terminus of KDO-8-P phosphatase is longer than KDN-9-P phosphatase, and partially overlaps the position of the sialic acid ring. The active site Mg²⁺ is depicted as a magenta sphere.