Abstract
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 beta-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 Mn(2+) and Ni(2+) for catalysis, whereas Zn(2+) 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 Mn(2+) ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn(2+) 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.
Highlights
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
PhnP is predicted based on its sequence to be a member of the -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 belong to the tRNase Z family [10, 11]
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
Summary
Phosphodiester bond near the 3Ј-end of pre-tRNA, yielding a 3Ј-end that can be coupled to an amino acid. These enzymes typically use two active site bound Zn2ϩ ions to simultaneously lower the pKa 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
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