Structural Basis for the Divergence of Substrate Specificity and Biological Function within HAD Phosphatases in Lipopolysaccharide and Sialic Acid Biosynthesis

Abstract

The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases, which, relative to members of other phosphatase families, have been particularly successful at adaptation to novel biological functions. Herein, we examine the structural basis for divergence of function in two bacterial homologs: 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 of 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 towards 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. Structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in active site structure that underlies 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; fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue, which 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, separated by diffuse active-site sequence boundaries. We infer high tolerance of the KDO8PP catalytic platform to amino acid replacements that, in turn, influence substrate specificity changes and thereby facilitate divergence of biological function.

Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1

“…Beyond affording higher atomic resolution than previous 2.1 Å SpFcp1 structures, the new AlF 3 structure captures the "correct" trigonal bi-pyramidal geometry of the transition state, rather than the square bi-pyramidal configuration of the earlier AlF 4 -complex (Ghosh et al 2008). The short bond distances from the aluminum center in AlF 3 to the apically positioned attacking water and aspartyl-Oδ leaving group weigh in favor of an associative mechanism of phosphoryl transfer by Fcp1, as has been observed for other members of the acylphosphatase superfamily (Wang et al 2002;Lahiri et al 2003;Lu et al 2008;Daughtry et al 2013). The AlF 3 structure highlights the relative "quietness" of the active site during progression from Michaelis complex to hydrolytic transition state, whereby none of the enzymic or Mg 2+ contacts to the scissile phosphate in the ground state are remodeled in the transition state.…”

Panoramic view of a superfamily of phosphatases through substrate profiling

“…The structure (PDB ID code: 2OBB) revealed a short C0 cap insert and crystal packing and standardized size-exclusion chromatography performed herein was consistent with a monomeric protein. HADSF enzymes with C0 cap types have been associated with histidine biosynthesis (30), inner core lipopolysaccharide biosynthesis (20), glycolipid and glycoprotein decoration for cell surface display (31,32), tRNA repair (33), and phosphoprotein phosphatase activity (23,34). As is the case with Q8A9J5 the HADSF polynucleotide phosphatases and phosphoprotein phosphatases are monomeric [in published cases Fcp1, Scp1, dullard, MDP-1, and T4 polynucleotide phosphatase/ kinase (23,(33)(34)(35)(36)], apparently allowing recognition of large polymeric (i.e., protein and nucleic acid) substrates.…”

Vanadium and proteins: Uptake, transport, structure, activity and function

“…In the 1.9 Å resolution structure of a C3 exoenzyme from Clostridium botulinum (PDB: 1UZI), Na 3 VO 4 was used in the crystallization medium and the formed tetravanadate and vanadate(V) ions participate in the crystal-packing stabilization [286]. Similarly, Na 3 VO 4 was also used during the crystallization procedures of a 2.8 Å resolution structure of a uridine [289] [290]. These studies, along with some other experimental approaches namely sitedirected mutagenesis, provided valuable information regarding the mechanism of action of the proteins elucidating the geometry of the complex and the residues involved in the transition state, as described in other sections of the this review.…”