Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the β-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(p-nitrophenyl)phosphate and 2′,3′-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn²⁺ and Ni²⁺ for catalysis, whereas Zn²⁺ afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn²⁺ ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn²⁺ binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.In many environments inorganic phosphate, an essential nutrient, can fall to extremely low concentrations, forcing microorganisms to utilize other forms of phosphorus to survive. In such cases, organophosphonates can comprise a major fraction of the total phosphorus available to biological systems (e.g. 2-aminoethylphosphonate is a widespread natural product). However, cleavage of the highly stable carbon-phosphorus (CP)⁵ bond to release inorganic phosphate requires specialized enzymes. One such enzyme activity found widely in bacteria is CP-lyase (1). Cleavage of the CP bond of organophosphonates by CP-lyase yields inorganic phosphate and, remarkably, a hydrocarbon. CP-lyase is actually a multienzyme system, encoded by the phn operon (phnCDEFGHIJKLMNOP), which is induced by low concentrations of phosphate as part of the pho regulon. Gene deletion studies in Escherichia coli have shown that phnGHIJKLM are essential for catalysis of CP bond cleavage, whereas the remaining genes probably encode transport, regulatory, or accessory functions (2). Only a handful of the proteins encoded by the phn operon have been characterized to date. PhnD was shown to be a periplasmic binding protein with high affinity for organophosphonates (3); the three-dimensional structure of PhnH, one of the proteins essential for CP-lyase catalysis, was recently solved, but a function has yet to be determined (4); PhnN was shown to be an ATP-dependent kinase that provides a redundant pathway to 5-phospho-d-ribofuranosyl-α-1-diphosphate (5); and PhnO was demonstrated to be an acetyl-CoA-dependent N-acyltransferase with activity toward a wide range of aminoalkylphosphonates (6).Although the phnP gene is not essential for CP bond cleavage by cells in liquid culture (2), cell growth on solid media supplemented with methylphosphonate or phosphite as the sole phosphorus source is prevented by phnP mutations (7), suggesting a critical regulatory or accessory role for PhnP. Accordingly, phnP appears frequently in the phn operon in various species of bacteria, typically following the phnN gene (8). PhnP is predicted based on its sequence to be a member of the β-lactamase family of metal-dependent hydrolases with greatest homology to enzymes from the tRNase Z (ProDom family PD352433) and ElaC families (9), the latter erroneously annotated as composed of arylsulfatases but later determined to also belong to the tRNase Z family (10, 11). The tRNase Z enzymes are endonucleases used by prokaryotes and eukaryotes to cleave a specific phosphodiester bond near the 3′-end of pre-tRNA, yielding a 3′-end that can be coupled to an amino acid. These enzymes typically use two active site bound Zn²⁺ ions to simultaneously lower the pK_a of a nucleophilic water molecule and stabilize negative charge development on the phosphodiester linkage undergoing nucleophilic attack (12). Since it is not clear how a tRNase activity would support cell growth with an organophosphonate as a sole phosphorus source, we set out to characterize the substrate specificity and three-dimensional structure of PhnP to learn more about this critical CP-lyase enzyme.