In order to accurately predict function, it is imperative that we understand how to distinguish between orthologous and paralogous enzymes. To address this, the HAD Bridging Project and the Structure Core experimentally characterized two paralogues, 2-keto-3-deoxy-D-manno-octulosonate 8-phosphate phosphohydrolase (KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9PP), from Bacteriodes thetaiotaomicron. These members of the HAD family catalyze phosphate ester hydrolysis in the pathway leading to lipid A and in the pathway leading to a capsular polysaccharide, respectively. Detailed structure/function analysis via a combination of kinetics, bioinformatics, and crystallography of liganded complexes was performed and allowed for specificity markers to be identified that otherwise would not have been obvious via simple “sequence-gazing.”
The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases that have been particularly successful at adaptating to novel biological functions relative to members of other phosphatase families. Herein, we examine the structural basis for the divergence of function in two bacterial homologues: 2-keto-3-deoxy-d-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step in KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities toward catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3(-) and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3(-) revealed the substrate-binding residues. The structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in their active-site structures that underlie the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single-domain proteins or fused with the pathway nucleotidyl transferases; the fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue that forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover, independently evolved lineages of KDO8PP orthologs exist, and are separated by diffuse active-site sequence boundaries. We infer a high tolerance of the KDO8PP catalytic platform to amino acid replacements that in turn influence substrate specificity changes and thereby facilitate the divergence in biological function.
Figure 1. KD9PP monomer (A) and tetramer (B) structures (PDB ID 3E8M). The HADSF Rossmann-fold domain is colored gray or light blue, with the tetramerization flap colored yellow. The HADSF catalytic motifs are colored red (motif 1), green (motif 2), cyan (motif 3), and orange (motif 4), and the magnesium cofactor is shown in magenta. This figure and all others unless indicated were generated with MOLSCRIPT(41) and POVscript+.(42)
Figure 2. Structure of BT-KDN9PP-Mg2+-VO3–-KDN. (A) Dimer from tetrameric BT-KDN9PP. The catalytic subunit is colored dark blue, and the cap subunit, light blue. The HAD catalytic motifs are shown as dark-blue sticks, and KDN, as yellow sticks. Vanadium is colored slate blue, and magnesium is a magenta sphere. (B) HAD catalytic residues and (C) KDN-binding residues. Panels B and C are colored the same as in panel A.
Figure 3. Structure of HI-KDO8PP-Mg2+-VO3–-KDO. (A) Dimer of HI-KDO8PP (generated from symmetry mates comprising tetramer). The catalytic subunit is colored forest green, and the cap subunit, light green. The HAD catalytic motifs are shown as forest-green sticks, and KDN, as yellow sticks. Vanadium is colored slate blue, and magnesium is a magenta sphere. (B) HAD catalytic residues and (C) KDN-binding residues. Panels B and C are colored the same as in panel A.
Figure 4. Overlay of the HI-KDO8PP and BT-KDO8PP active sites. The dimer of HI-KDO8PP (generated from symmetry mates comprising tetramer) with its catalytic subunit colored forest green, and cap subunit, light green. KDN is shown as yellow sticks, vanadium is colored slate blue, and magnesium is a magenta sphere. The dimer of BT-KDO8PP with its catalytic subunit colored dark orange, and cap subunit, light orange. Magnesium is an orange sphere. The putative substrate-binding residues are shown as sticks and labeled in their corresponding enzyme color. Hydrogen bonds and coordination bonds are shown as dashed lines.
Figure 5. Schematics of Bioinformatic Analyses. (A) Tree of life generated from iTOL (itol.embl.de) displaying phyla (and class in the case of proteobacteria) that contain KDO8PP and KDN9PP sequences. Phyla are color-coded (KDO8PP blue and KDN9PP green) on the basis of the majority presence of enzymes. Red boxes indicate the two phyla that contain species with both KDN9PP and KDO8PP. The type of KDO8PP enzyme (Arg, Lys, or Gly) is indicated next to the phyla name. (B) Unrooted phylogenetic tree generated using a multiple-sequence alignment of all KDO8PP and KDN9PP enzymes identified herein. KDO8PP-Arg enzymes are colored blue, KDO8PP-Gly, green, KDN9PP, light orange, and KDN9PP-KDN–CMP transferase fusions, deep orange. Red stars indicate enzymes that were identified where an additional KDN9PP or KDO8PP gene exists within the species. Black symbols (#) indicate KDO8PP-Arg-KDO–CMP transferase fusion genes. The phyla of some KDO8PP-Gly enzymes have been labeled. This figure was generated with FigTree (http://tree.bio.ed.ac.uk/software/figtree/).
Reprinted with permission from Biochemistry. Copyright 2013 American Chemical Society.