Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

Abstract

SUMMARY Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 Å resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases, but with notable differences such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional co-crystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently-bound transition state mimic, provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

Vanadium and proteins: Uptake, transport, structure, activity and function

“…Following a PhnA structure from atoms are directly hydrogen bonded to the protein residues. In the same work, PhnA was also crystallized in the presence of substrate and one of the variants T68A, clarifying the importance of these residues and the geometrical constrains crucial for the hydrolysis [446].…”

“…The PhnA active site comprises two Zn 2+ ions, similar to alkaline phosphatases, coordinated to His215, His377 and Asp211, for metal 1 (M1), and Asp29, Asp250, and His251 in the case of metal 2 (M2). The metals are 4.6 Å apart and, even though other divalent cations can be incorporated and promote catalysis, Sinorhizobium meliloti PhnA has higher specificity towards zinc[446].For catalysis, phosphonoacetate sits in the active site, making hydrogen bond interactions with the two zinc ions (M1 and M2). The 3T00 structure represents the transition state during catalysis and a vanadate(V) moiety is located in the active site[446].…”

“…The metals are 4.6 Å apart and, even though other divalent cations can be incorporated and promote catalysis, Sinorhizobium meliloti PhnA has higher specificity towards zinc[446].For catalysis, phosphonoacetate sits in the active site, making hydrogen bond interactions with the two zinc ions (M1 and M2). The 3T00 structure represents the transition state during catalysis and a vanadate(V) moiety is located in the active site[446]. Vanadate(V) presents a distorted trigonal-bipyramidal geometry, interacting with the two metal sites via the oxygen ligands.…”

“…Pseudomonas fluorescens (PDB: 1EI6)[445], other similar structures from Sinorhizobium meliloti 1021 were obtained including a PhnA complexed with vanadate(V) structure at 1.8 Å resolution (PDB: 3T00)[446]. The PhnA active site comprises two Zn 2+ ions, similar to alkaline phosphatases, coordinated to His215, His377 and Asp211, for metal 1 (M1), and Asp29, Asp250, and His251 in the case of metal 2 (M2).…”

Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

“…Alternatively, phosphonoacetaldehyde is oxidized to phosphonoacetate catalyzed by phosphonoacetaldehyde dehydrogenase (59). Also, hydrolytic activities for phosphonoacetate (59,(86)(87)(88)(89)(90) as well as for phosphonopyruvate (91) have been described, and a hydrolytic activity for phosphonoformaldehyde has been suggested (21). Phosphonopyruvate in turn may be formed by the activities of phosphoenolpyruvate phosphomutase or phosphoalanine:2-oxoglutarate transaminase (91)(92)(93).…”