The catalytic scaffold of the haloalkanoic acid dehalogenase enzyme superfamily acts as a mold for the trigonal bipyramidal transition state

Abstract

The evolution of new catalytic activities and specificities within an enzyme superfamily requires the exploration of sequence space for adaptation to a new substrate with retention of those elements required to stabilize key intermediates/transition states. Here, we propose that core residues in the large enzyme family, the haloalkanoic acid dehalogenase enzyme superfamily (HADSF) form a ''mold'' in which the trigonal bipyramidal transition states formed during phosphoryl transfer are stabilized by electrostatic forces. The vanadate complex of the hexose phosphate phosphatase BT4131 from Bacteroides thetaiotaomicron VPI-5482 (HPP) determined at 1.00 Å resolution via X-ray crystallography assumes a trigonal bipyramidal coordination geometry with the nucleophilic Asp-8 and one oxygen ligand at the apical position. Remarkably, the tungstate in the complex determined to 1.03 Å resolution assumes the same coordination geometry. The contribution of the general acid/base residue Asp-10 in the stabilization of the trigonal bipyramidal species via hydrogen-bond formation with the apical oxygen atom is evidenced by the 1.52 Å structure of the D10A mutant bound to vanadate. This structure shows a collapse of the trigonal bipyramidal geometry with displacement of the water molecule formerly occupying the apical position. Furthermore, the 1.07 Å resolution structure of the D10A mutant complexed with tungstate shows the tungstate to be in a typical ''phosphate-like'' tetrahedral configuration. The analysis of 12 liganded HADSF structures deposited in the protein data bank (PDB) identified stringently conserved elements that stabilize the trigonal bipyramidal transition states by engaging in favorable electrostatic interactions with the axial and equatorial atoms of the transferring phosphoryl group.phosphatase ͉ phosphoryl transfer ͉ transition state stabilization ͉ trigonal bipyramidal phosphorane

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.…”

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…The role of the metal cofactor is generally to bind the phosphate group of the substrate, correctly orientating the phosphorus atom for nucleophilic attack. In the phosphatases of the large haloacid dehydrogenase family, attack by the Asp nucleophile leads to the formation of an aspartylphosphate intermediate, which is hydrolyzed by nucleophilic attack of a water molecule (58,59). If a similar mechanism were to operate in the colicin M-like bacteriocins, Asp-235, which has been shown to be essential for catalytic activity, would be a likely candidate for the attacking nucleophile (41).…”

Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF3- rather than by phosphoranes

“…mechanical -molecular mechanical (QM-MM) analysis based on the structure (19) and was subsequently validated in solution by direct observation based on 19 F NMR data (8). However, the MgF − 3 interpretation was questioned (20,21) and the pentacovalent phosphorane interpretation defended (22,23), despite the presence of fluoride, which severely compromises the catalytic cycle of β-PGM (15). Therefore, using an integration of NMR and high-resolution x-ray structural data, we now report a thorough characterization of β-PGM in the presence of G6P and fluoride that leaves no room for a pentacovalent phosphorane interpretation of solid-state or solution-state complexes.…”