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Literature summary extracted from

  • Chen, E.; Choy, M.S.; Petrenyi, K.; Konya, Z.; Erdoedi, F.; Dombradi, V.; Peti, W.; Page, R.
    Molecular insights into the fungus-specific serine/threonine protein phosphatase Z1 in Candida albicans (2016), mBio, 7, e00872-16 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.1.3.16 gene PPZ1, recombinant expression of N-terminally GST-tagged enzyme or His6-tagged catalytic subunit PPZ1cat and His6-tagged N-terminal IDP domain PPZ1FL in Escherichia coli strain BL21(DE3) RIL cells Candida albicans

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.1.3.16 purified recombinant detagged apo-form of the catalytic subunit, apo-PPZ1cat, or fused to microcystin-LR (the marine toxin microcystin-LR is a potent cyclic peptide inhibitor of PP1), by hanging drop vapor diffusion method, mixing of enzyme in 20 mM Tris, pH 8.0, 500 mM NaCl, 0.5 mM Tris(2-carboxyethyl) phosphine (TCEP), and 1 mM MnCl21.8 M with ammonium citrate tribasic, pH 7.0, in a 1:1 ratio, and subsequent mixture in a 2:1 protein/crystallization condition ratio with reservoir solution containing 0.06 M citric acid, pH 4.1, and 16% w/v PEG 3350, X-ray diffraction structure determination and analysis at 2.4-2.6 A resolution, molecular replacement using PP1 stucture, PDB ID 4MOV, as the search model Candida albicans

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.1.3.16 I-2 inhibitor-2, an inhibitory regulator from rabbit skeletal muscle. I-2 is a much less potent inhibitor against PPZ1FL, which includes the about 160-amino-acid N-terminal IDP domain, as compared to the catalytic domain. The N-terminal domain likely masks one or more binding sites for I-2 but not the active site, as the PPZ1FL protein is fully susceptible to microcystin-LR inhibition and prevents PPZ1 inhibition by this protein inhibitor Candida albicans
3.1.3.16 microcystin-LR i.e. MC, the marine toxin microcystin-LR is a potent cyclic peptide inhibitor of PP1, it binds the active site of PPZ1cat, binding structure analysis, overview. The bulk of the MC binds the PPZ1cat hydrophobic binding pocket, while the remainder covers and, as a consequence, blocks the active site Candida albicans

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.1.3.16 additional information Candida albicans the minimal PP1-binding domains of regulators GADD34, PNUTS, and spinophilin bind PP1x02with strong affinities. The catalytic domain PPZ1cat binds GADD34 less effectively, as compared to the phosphatase PP1, largely due to an inability of GADD34552-567 to productively bind the PhiPhi motif binding pocket, pulldown assays, overview. The altered PhiPhi binding pocket and the presence of the Z1-helix negatively impact the binding of the regulators to PPZ1. PPZ1, compared to PP1alpha, does not effectively pull down either PNUTS or spinophilin (about 85% less binding) ?
-
 ?
3.1.3.16 [a protein]-serine/threonine phosphate + H2O Candida albicans
-
 [a protein]-serine/threonine + phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.1.3.16 Candida albicans D5HRC1
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.1.3.16 recombinant GST-tagged enzyme PPZ1 from Escherichia coli strain BL21(DE3) RIL cells by glutathione affinity chromatography in presence of Mn2+ ions, the GST tag is removed using the PreScission protease during elution. Recombinant His6-tagged catalytic subunit PPZ1cat and His6-tagged N-terminal IDP domain PPZ1FL from Escherichia coli strain BL21(DE3) RIL cells by nickel affinity chromatography and gel filtration, the His6-tag is cleaved off by tobacco etch virus (TEV) protease, followed by gel filtration Candida albicans

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.1.3.16 0.0046
-
 purified recombinant N-terminal IDP domain PPZ1FL, pH 8.0, 25°C Candida albicans
3.1.3.16 1.2
-
 purified recombinant catalytic domain PPZ1cat, pH 8.0, 25°C Candida albicans

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.3.16 additional information the minimal PP1-binding domains of regulators GADD34, PNUTS, and spinophilin bind PP1x02with strong affinities. The catalytic domain PPZ1cat binds GADD34 less effectively, as compared to the phosphatase PP1, largely due to an inability of GADD34552-567 to productively bind the PhiPhi motif binding pocket, pulldown assays, overview. The altered PhiPhi binding pocket and the presence of the Z1-helix negatively impact the binding of the regulators to PPZ1. PPZ1, compared to PP1alpha, does not effectively pull down either PNUTS or spinophilin (about 85% less binding) Candida albicans ?
-
 ?
3.1.3.16 [a protein]-serine/threonine phosphate + H2O
-
 Candida albicans [a protein]-serine/threonine + phosphate
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.1.3.16 PPZ1
-
 Candida albicans
3.1.3.16 serine/threonine protein phosphatase
-
 Candida albicans

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.1.3.16 25
-
 assay at Candida albicans

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.1.3.16 8
-
 assay at Candida albicans

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
3.1.3.16 0.000007
-
 pH 8.0, 25°C, recombinant catalytic domain of PPZ1, PPZ1cat Candida albicans I-2
3.1.3.16 0.000278
-
 pH 8.0, 25°C, recombinant N-terminal IDP domain of PPZ1, PPZ1FL Candida albicans I-2

General Information

EC Number General Information Comment Organism
3.1.3.16 additional information the widened Z1-helix binding pocket results in the second significant structural difference between PPZ1cat and PP1alpha: the C-terminal residues of PPZ1cat are not disordered like they are in PP1alpha, but instead form an alpha-helix that nestles into this widened Z1-helix binding pocket. This interaction is stabilized by hydrophobic interactions between the C-terminal helix and residues from helices A' and B, with the interaction centered on PPZ1 C-terminal helix residue Met473PPZ1. This residue is completely buried from solvent via interactions with Leu464PPZ1, Leu469PPZ1, and Val472PPZ1 from the C-terminal helix as well as by interactions with Phe185PPZ1 from helix A' and His235PPZ1, Ile238PPZ1, and Arg239PPZ1 from helix B. The PPZ1-specific helix is dynamic. PPZ1 binds only a subset of PP1 regulatory proteins Candida albicans