Structure and Kinetics of Phosphonopyruvate Hydrolase from Voriovorax sp. Pa12: New Insight into the Divergence of Catalysis within the PEP Mutase/Isocitrate Lyase Superfamily

• Published in: Biochemistry (Easton) 2006-09, Vol.45 (38), p.11491-11504
• Main Authors: Chen, CCH, Han, Y, Niu, W, Kulakova, AN, Howard, A, Quinn, J P, Dunaway-Mariano, D, Herzberg, O
• Format: Article
• Language: English
• Subjects: Escherichia coli; Variovorax
• ISSN: 0006-2960
• DOI: 10.1021/bi0612081

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. Pa12 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 Mg super(+2) binding first (K sub(d) =140 plus or minus 40 mu M), are k sub(cat) = 105 plus or minus 2 s super(-1) and P-pyr K sub(m) = 5 plus or minus 1 mu M. PEP (slow substrate k sub(cat) = 2 x 10 super(-4) s super(-1)), oxalate, and sulfopyruvate are competitive inhibitors with K sub(i) values of 2.0 plus or minus 0.1 mM, 17 plus or minus 1 mu M, and 210 plus or minus 10 mu M, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg super(2+) and oxalate (inhibitor), and the enzyme bound to Mg super(2+) 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 ) sub(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 Mg super(2+). 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.