Recombinant Variovorax sp. Phosphonopyruvate hydrolase (pphA)

Enzyme Classification and Biological Role

Recombinant PPH from Variovorax sp. Pal2 is a Mg²⁺-dependent hydrolase that catalyzes the conversion of phosphonopyruvate to pyruvate and inorganic phosphate . It plays a vital role in global phosphorus cycling by degrading phosphonates, which are naturally occurring compounds with C-P bonds resistant to typical hydrolytic enzymes . Unlike other C-P bond-cleaving enzymes (e.g., phosphonoacetaldehyde hydrolase), PPH is evolutionarily linked to the PEPM/ICL superfamily, sharing 41% sequence identity with PEP mutase from Tetrahymena pyriformis .

Quaternary Structure

• Oligomerization: Analytical gel filtration reveals PPH exists as a tetramer in solution, with each monomer contributing to the active site architecture .

• Fold: Each monomer adopts an (α/β)₈ barrel fold, with the eighth helix swapped between two subunits in the dimer-of-dimers assembly .

Active Site Features

• Metal Coordination: Mg²⁺ anchors substrates (P-pyr) and inhibitors (oxalate) via interactions with conserved residues (e.g., Asp42, Thr118) .

• Flexible Gating Loop: A disordered loop (residues 112–121) near the active site contrasts with PEPM, which adopts ordered conformations during catalysis .

Kinetic Properties

Steady-state kinetic analyses reveal a rapid equilibrium ordered mechanism:

Inhibitors

• Oxalate: Competitive inhibitor (K_i = 17 ± 1 μM) .

• Sulfopyruvate: K_i = 210 ± 10 μM .

• Phosphoenolpyruvate (PEP): Acts as a slow substrate (k_cat = 2 × 10⁻⁴ s⁻¹) .

Catalytic Mechanism

The proposed mechanism involves:

1. Mg²⁺ Binding: Primes the active site for substrate coordination .

2. Nucleophilic Attack: A water molecule activated by Thr118 attacks the phosphorus atom of P-pyr, cleaving the C-P bond .

3. Product Release: Pyruvate and phosphate dissociate, with the gating loop remaining disordered throughout the cycle .

This mechanism diverges from PEPM, which uses a conserved lysine for substrate stabilization .

Biotechnological Applications

• Environmental Remediation: Degrades phosphonates in soil and marine ecosystems, reducing anthropogenic phosphonate pollution .

• Enzyme Engineering: Structural insights (e.g., PDB ID: 2DUA) aid in designing variants for industrial biocatalysis .

Comparative Analysis with Related Enzymes

Expression and Purification

• Host System: Recombinant PPH is expressed in Escherichia coli with high yield (~85% purity via SDS-PAGE) .

• Stability: Retains activity under broad pH and temperature ranges, making it suitable for industrial processes .