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Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2

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Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2. / Roberts, SJ; Stewart, Alan James; Schmid, R; Blindauer, CA; Bond, SR; Sadler, PJ; Farquharson, C.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1752, No. 1, 31.08.2005, p. 73-82.

Research output: Contribution to journalArticlepeer-review

Harvard

Roberts, SJ, Stewart, AJ, Schmid, R, Blindauer, CA, Bond, SR, Sadler, PJ & Farquharson, C 2005, 'Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2', Biochimica et Biophysica Acta - Proteins and Proteomics, vol. 1752, no. 1, pp. 73-82. https://doi.org/10.1016/j.bbapap.2005.06.009

APA

Roberts, SJ., Stewart, A. J., Schmid, R., Blindauer, CA., Bond, SR., Sadler, PJ., & Farquharson, C. (2005). Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2. Biochimica et Biophysica Acta - Proteins and Proteomics, 1752(1), 73-82. https://doi.org/10.1016/j.bbapap.2005.06.009

Vancouver

Roberts SJ, Stewart AJ, Schmid R, Blindauer CA, Bond SR, Sadler PJ et al. Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2. Biochimica et Biophysica Acta - Proteins and Proteomics. 2005 Aug 31;1752(1):73-82. https://doi.org/10.1016/j.bbapap.2005.06.009

Author

Roberts, SJ ; Stewart, Alan James ; Schmid, R ; Blindauer, CA ; Bond, SR ; Sadler, PJ ; Farquharson, C. / Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2. In: Biochimica et Biophysica Acta - Proteins and Proteomics. 2005 ; Vol. 1752, No. 1. pp. 73-82.

Bibtex - Download

@article{995889b4779b4c52b383febe2118e167,
title = "Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2",
abstract = "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 (Vmax of 633 nmol min−1 mg−1 and Km of 45.5 μM). 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.",
keywords = "Bone mineralization, Haloacid dehalogenase superfamily, Molecular modelling, Phosphatase, Site-directed mutagenesis",
author = "SJ Roberts and Stewart, {Alan James} and R Schmid and CA Blindauer and SR Bond and PJ Sadler and C Farquharson",
note = "Partially funded by BBSRC and the Welcome Trust",
year = "2005",
month = aug,
day = "31",
doi = "10.1016/j.bbapap.2005.06.009",
language = "English",
volume = "1752",
pages = "73--82",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "1",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2

AU - Roberts, SJ

AU - Stewart, Alan James

AU - Schmid, R

AU - Blindauer, CA

AU - Bond, SR

AU - Sadler, PJ

AU - Farquharson, C

N1 - Partially funded by BBSRC and the Welcome Trust

PY - 2005/8/31

Y1 - 2005/8/31

N2 - 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 (Vmax of 633 nmol min−1 mg−1 and Km of 45.5 μM). 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.

AB - 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 (Vmax of 633 nmol min−1 mg−1 and Km of 45.5 μM). 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.

KW - Bone mineralization

KW - Haloacid dehalogenase superfamily

KW - Molecular modelling

KW - Phosphatase

KW - Site-directed mutagenesis

U2 - 10.1016/j.bbapap.2005.06.009

DO - 10.1016/j.bbapap.2005.06.009

M3 - Article

VL - 1752

SP - 73

EP - 82

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 1

ER -

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