Structure and Kinetics of Phosphonopyruvate Hydrolase from Voriovorax sp. Pal2:  New Insight into the Divergence of Catalysis within the PEP Mutase/Isocitrate Lyase Superfamily^,

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.

Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily,

“…This appears to be a significant distortion but should be taken with caution because the oxalate crystallographic temperature factors are relatively high at 42 Å 2 . The mode of oxalate interaction with the protein groups is the same as observed for the crystal structures of PEP mutase (7) and phosphonopyruvate hydrolase (5), and comparable to the mode of pyruvate interaction with protein groups observed for the crystal structure of 2-methylisocitrate lyase (1). The arginine, oxyanion hole and the Mg +2 binding residues comprise the core residues of the superfamily catalytic scaffold.…”

Depth distributions of alkaline phosphatase and phosphonate utilization genes in the North Pacific Subtropical Gyre

“…Only the top hit known as PhnA or PhnX was treated as the true homolog. PalA is another C-P hydrolase; it resembles phosphoenolpyruvate mutase (PEPM, an enzyme responsible for C-P bond synthesis) at the sequence and structural level (Chen et al 2006), and both proteins are annotated as PEPM in CDD. PalA is distinguished from PEPM by the presence of Thr118 in the mobile loop (Chen et al 2006).…”

“…PalA is another C-P hydrolase; it resembles phosphoenolpyruvate mutase (PEPM, an enzyme responsible for C-P bond synthesis) at the sequence and structural level (Chen et al 2006), and both proteins are annotated as PEPM in CDD. PalA is distinguished from PEPM by the presence of Thr118 in the mobile loop (Chen et al 2006). It is important to note that in some PalA peptides this Thr is replaced by Asn, which is used by PEPM, making it indistinguishable from PEPM in this case.…”

Diversity and abundance of phosphonate biosynthetic genes in nature

“…We searched for pepM homologs in the Integrated Microbial Genomes (IMG) database (39), GOS marine metagenomes (35,36), and the Integrated Microbial Genomes with Microbiome Samples (IMG/M) database (40), using the sequences of biochemically validated PepM sequences as queries (31,33,34). All potential hits were screened for the highly conserved catalytic motif EDKXXXXXNS, which distinguishes PEP mutase from other members of the isocitrate lyase superfamily (41).…”