2DUA

Crystal Structure of Phosphonopyruvate Hydrolase Complex with Oxalate and Mg++

Summary for 2DUA

• Entry DOI: 10.2210/pdb2dua/pdb
• Related: 2HJP 2HRW
• Descriptor: Phosphonopyruvate hydrolase, alpha-D-xylopyranose, OXALATE ION, ... (7 entities in total)
• Functional Keywords: phosphonopyruvate hydrolase, phosphonopyruvate, phosphorus-carbon bond cleavage, pep mutase/isocitrate lyase superfamily, hydrolase
• Biological source: Variovorax sp.
• Total number of polymer chains: 1
• Total formula weight: 31572.08

Authors

Herzberg, O.,Chen, C. (deposition date: 2006-07-21, release date: 2006-10-03, Last modification date: 2024-02-14)

Primary citation

Chen, C.C.H.,Han, Y.,Niu, W.,Kulakova, A.N.,Howard, A.,Quinn, J.P.,Dunaway-Mariano, D.,Herzberg, O.
Structure and Kinetics of Phosphonopyruvate Hydrolase from Voriovorax sp. Pal2: New Insight into the Divergence of Catalysis within the PEP Mutase/Isocitrate Lyase Superfamily
Biochemistry, 45:11491-11504, 2006

PubMed Abstract: Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range. The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg2+ binding first (Kd = 140 +/- 40 microM), are kcat = 105 +/- 2 s(-1) and P-pyr Km = 5 +/- 1 microM. PEP (slow substrate kcat = 2 x 10(-4) s(-1)), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 +/- 0.1 mM, 17 +/- 1 microM, and 210 +/- 10 microM, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (alpha/beta)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.

PubMed: 16981709
DOI: 10.1021/bi061208l

Experimental method

X-RAY DIFFRACTION (2 Å)