Publications

• Phosphorylcholine phosphatase: a peculiar enzyme of Pseudomonas aeruginosa.

  Domenech C.E., Otero L.H., Beassoni P.R., Lisa A.T.

  Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg(2+) or Zn(2+), PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expres ... >> More

  Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg(2+) or Zn(2+), PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues (42)E, (43)E and (82)YYY(84). Zn(2+) is better activator than Mg(2+) at pH 5.0 and it is more effective at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn(2+) induces at pH 5.0 a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. However, at pH 7.4, this effect is not observed because to the hydrolysis of the [Zn(2+)L(2) (-1)L(2) (0)(H(2)O)(2)] complex, which causes a change from octahedral to tetrahedral in the metal coordination geometry. This enzyme is also present in P. fluorescens, P. putida, P. syringae, and other organisms. We have recently crystallized PchP and solved its structure. << Less

  Enzyme Res. 2011:561841-561841(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Human PHOSPHO1 exhibits high specific phosphoethanolamine and phosphocholine phosphatase activities.

  Roberts S.J., Stewart A.J., Sadler P.J., Farquharson C.

  Human PHOSPHO1 is a phosphatase enzyme for which expression is upregulated in mineralizing cells. This enzyme has been implicated in the generation of P(i) for matrix mineralization, a process central to skeletal development. PHOSPHO1 is a member of the haloacid dehalogenase (HAD) superfamily of M ... >> More

  Human PHOSPHO1 is a phosphatase enzyme for which expression is upregulated in mineralizing cells. This enzyme has been implicated in the generation of P(i) for matrix mineralization, a process central to skeletal development. PHOSPHO1 is a member of the haloacid dehalogenase (HAD) superfamily of Mg2+-dependent hydrolases. However, substrates for PHOSPHO1 are, as yet, unidentified and little is known about its activity. We show here that PHOSPHO1 exhibits high specific activities toward phosphoethanolamine (PEA) and phosphocholine (PCho). Optimal enzymic activity was observed at approx. pH 6.7. The enzyme shows a high specific Mg2+-dependence, with apparent K(m) values of 3.0 microM for PEA and 11.4 microM for PCho. These results provide a novel mechanism for the generation of P(i) in mineralizing cells from PEA and PCho. << Less

  Biochem. J. 382:59-65(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2.

  Roberts S.J., Stewart A.J., Schmid R., Blindauer C.A., Bond S.R., Sadler P.J., Farquharson C.

  PHOSPHO1, a phosphoethanolamine/phosphocholine phosphatase, is upregulated in mineralising cells and is thought to be involved in the generation of inorganic phosphate for bone mineralisation. PHOSPHO2 is a putative phosphatase sharing 42% sequence identity with PHOSPHO1. Both proteins contain thr ... >> More

  PHOSPHO1, a phosphoethanolamine/phosphocholine phosphatase, is upregulated in mineralising cells and is thought to be involved in the generation of inorganic phosphate for bone mineralisation. PHOSPHO2 is a putative phosphatase sharing 42% sequence identity with PHOSPHO1. Both proteins contain three catalytic motifs, conserved within the haloacid dehalogenase superfamily. Mutation of Asp32 and Asp203, key residues within two motifs, abolish PHOSPHO1 activity and confirm it as a member of this superfamily. We also show that Asp43 and Asp123, residues that line the substrate-binding site in our PHOSPHO1 model, are important for substrate hydrolysis. Further comparative modelling reveals that the active sites of PHOSPHO1 and PHOSPHO2 are very similar, but surprisingly, recombinant PHOSPHO2 hydrolyses phosphoethanolamine and phosphocholine relatively poorly. Instead, PHOSPHO2 shows high specific activity toward pyridoxal-5-phosphate (V(max) of 633 nmol min(-1) mg(-1) and K(m) of 45.5 microM). Models of PHOSPHO2 and PHOSPHO1 suggest subtle differences in the charge distributions around the putative substrate entry site and in the location of potential H-bond donors. << Less

  Biochim. Biophys. Acta 1752:73-82(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes.

  Houston B., Seawright E., Jefferies D., Hoogland E., Lester D., Whitehead C., Farquharson C.

  Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differe ... >> More

  Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes. << Less

  Biochim. Biophys. Acta 1448:500-506(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Comparative modelling of human PHOSPHO1 reveals a new group of phosphatases within the haloacid dehalogenase superfamily.

  Stewart A.J., Schmid R., Blindauer C.A., Paisey S.J., Farquharson C.

  PHOSPHO1 is a recently identified phosphatase whose expression is upregulated in mineralizing cells and is implicated in the generation of inorganic phosphate for matrix mineralization, a process central to skeletal development. The enzyme is a member of the haloacid dehalogenase (HAD) superfamily ... >> More

  PHOSPHO1 is a recently identified phosphatase whose expression is upregulated in mineralizing cells and is implicated in the generation of inorganic phosphate for matrix mineralization, a process central to skeletal development. The enzyme is a member of the haloacid dehalogenase (HAD) superfamily of magnesium-dependent hydrolases. However, the natural substrate(s) is as yet unidentified and to date no structural information is known. We have identified homologous proteins in a number of species and have modelled human PHOSPHO1 based upon the crystal structure of phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The model includes the catalytic Mg(2+) atom bound via three conserved Asp residues (Asp32, Asp34 and Asp203); O-ligands are also provided by a phosphate anion and two water molecules. Additional residues involved in PSP-catalysed hydrolysis are conserved and are located nearby, suggesting both enzymes share a similar reaction mechanism. In PHOSPHO1, none of the PSP residues that confer the enzyme's substrate specificity (Arg56, Glu20, Met43 and Phe49) are conserved. Instead, we propose that two fully conserved Asp residues (Asp43 and Asp123), not present in PSPs contribute to substrate specificity in PHOSPHO1. Our findings show that PHOSPHO1 is not a member of the subfamily of PSPs but belongs to a novel, closely related enzyme group within the HAD superfamily. << Less

  Protein Eng. 16:889-895(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.