Expression, purification and preliminary diffraction studies of PhnP

Podzelinska, Kateryna; He, Shikun; Soares, Alexei S.; Zechel, David L.; Hove‐Jensen, Bjarne; Jia, Zongchao

doi:10.1107/s1744309108014656

Cited by 8 publications

(11 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PhnP was purified as previously described. 29 NMR spectra were recorded on Bruker Avance 400 or 600 MHz spectrometers. 1 H chemical shifts (δ) are reported relative to HDO, whereas 31 P chemical shifts are reported relative to a 17 mM phosphoric acid external standard.…”

Section: ' Discussionmentioning

confidence: 99%

“…α- d -Ribosyl 1-phosphate was prepared enzymatically by phosphorolysis of uridine with uridine phosphorylase in the presence of P i followed by ion-exchange chromatography on a Dowex 1 column by K. F. Jensen, University of Copenhagen. PhnP was purified as previously described . NMR spectra were recorded on Bruker Avance 400 or 600 MHz spectrometers.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Physiological Role of phnP-specified Phosphoribosyl Cyclic Phosphodiesterase in Catabolism of Organophosphonic Acids by the Carbon−Phosphorus Lyase Pathway

2011

Self Cite

View full text Add to dashboard Cite

In Escherichia coli , internalization and catabolism of organophosphonicacids are governed by the 14-cistron phnCDEFGHIJKLMNOP operon. The phnP gene product was previously shown to encode a phosphodiesterase with unusual specificity toward ribonucleoside 2',3'-cyclic phosphates. Furthermore, phnP displays shared synteny with phnN across bacterial phn operons. Here the role of PhnP was examined by (31)P NMR spectrometry on the culture supernatants of E. coli phn mutants grown in the presence of alkylphosphonic acid or phosphite. The addition of any of these alkylphosphonic acids or phosphite resulted in the accumulation of α-D-ribosyl 1,2-cyclic phosphate and α-D-ribosyl 1-alkylphosphonate in a phnP mutant strain. Additionally, α-D-ribosyl 1-ethylphosphonate was observed to accumulate in a phnJ mutant strain when it was fed ethylphosphonic acid. Purified PhnP was shown to regiospecifically convert α-D-ribosyl 1,2-cyclic phosphate to α-D-ribosyl 1-phosphate. Radiolabeling studies revealed that 5-phospho-α-D-ribosyl 1,2-cyclic phosphate also accumulates in a phnP mutant. This compound was synthesized and shown to be regiospecifically converted by PhnP to α-D-ribosyl 1,5-bisphosphate. It is also shown that organophosphonate catabolism is dependent on the synthesis of 5-phospho-α-D-ribosyl 1-diphosphate, suggesting that this phosphoribosyl donor is used to initiate the carbon-phosphorus (CP) lyase pathway. The results show that 5-phospho-α-D-ribosyl 1,2-cyclic phosphate is an intermediate of organophosphonic acid catabolism, and it is proposed that this compound derives from C-P bond cleavage of 5-phospho-α-D-ribosyl 1-alkylphosphonates by CP lyase.

show abstract

Section: ' Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Physiological Role of phnP-specified Phosphoribosyl Cyclic Phosphodiesterase in Catabolism of Organophosphonic Acids by the Carbon−Phosphorus Lyase Pathway

2011

Self Cite

View full text Add to dashboard Cite

show abstract

“…Expression, Purification, and Size Exclusion Chromatography of PhnP and Mutants-The cloning and expression of phnP as well as the purification and crystallization of wild type PhnP were described previously (13). Briefly, wild type PhnP with a C-terminal His 6 tag was expressed from the plasmid pHO520-phnP in BL21(DE3) cells (Novagen) co-transformed with pLacI (Novagen).…”

Section: Methodsmentioning

confidence: 99%

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

Podzelinska

Wathier

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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 ␤-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(pnitrophenyl)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 Å 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.

show abstract

“…The latter scenario is quite plausible, as PhnP containing ∼0.13 manganese ion per monomer was crystallized with a dinuclear active site (suggesting selective crystallization of the dinuclear enzyme). 26,41 In any case, PhnP is clearly catalytically competent and achieves its greatest activity in the presence of excess manganese ion where a dinuclear active site is expected, and indeed observed in the crystal structure generated in the presence of excess manganese ion. Interestingly, E. coli possesses a manganese ion sensory protein, MntR, that regulates the expression of mntH, encoding a transporter for manganese ion uptake 74 that can increase cytoplasmic manganese ion concentrations to >300 μM (and under certain conditions to 1−3 mM).…”

Section: Methodsmentioning

confidence: 83%

Structure and Mechanism of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway

He¹,

Wathier²,

Podzelinska³

et al. 2011

Biochemistry

Self Cite

View full text Add to dashboard Cite

PhnP is a phosphodiesterase that plays an important role within the bacterial carbon-phosphorus lyase (CP-lyase) pathway by recycling a "dead-end" intermediate, 5-phospho-α-d-ribosyl 1,2-cyclic phosphate, that is formed during organophosphonate catabolism. As a member of the metallo-β-lactamase superfamily, PhnP is most homologous in sequence and structure to tRNase Z phosphodiesterases. X-ray structural analysis of PhnP complexed with orthovanadate to 1.5 Å resolution revealed this inhibitor bound in a tetrahedral geometry by the two catalytic manganese ions and the putative general acid residue H200. Guided by this structure, we probed the contributions of first- and second-sphere active site residues to catalysis and metal ion binding by site-directed mutagenesis, kinetic analysis, and ICP-MS. Alteration of H200 to alanine resulted in a 6-33-fold decrease in k(cat)/K(M) with substituted methyl phenylphosphate diesters with leaving group pK(a) values ranging from 4 to 8.4. With bis(p-nitrophenyl)phosphate as a substrate, there was a 10-fold decrease in k(cat)/K(M), primarily the result of a large increase in K(M). Moreover, the nickel ion-activated H200A PhnP displayed a bell-shaped pH dependence for k(cat)/K(M) with pK(a) values (pK(a1) = 6.3; pK(a2) = 7.8) that were comparable to those of the wild-type enzyme (pK(a1) = 6.5; pK(a2) = 7.8). Such modest effects are counter to what is expected for a general acid catalyst and suggest an alternate role for H200 in this enzyme. A Brønsted analysis of the PhnP reaction with a series of substituted phenyl methyl phosphate esters yielded a linear correlation, a β(lg) of -1.06 ± 0.1, and a Leffler α value of 0.61, consistent with a synchronous transition state for phosphoryl transfer. On the basis of these data, we propose a mechanism for PhnP.

show abstract

Expression, purification and preliminary diffraction studies of PhnP

Cited by 8 publications

References 25 publications

Physiological Role of phnP-specified Phosphoribosyl Cyclic Phosphodiesterase in Catabolism of Organophosphonic Acids by the Carbon−Phosphorus Lyase Pathway

Physiological Role of phnP-specified Phosphoribosyl Cyclic Phosphodiesterase in Catabolism of Organophosphonic Acids by the Carbon−Phosphorus Lyase Pathway

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

Structure and Mechanism of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway

Contact Info

Product

Resources

About