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Literature summary extracted from

  • van Staalduinen, L.M.; McSorley, F.R.; Schiessl, K.; Seguin, J.; Wyatt, P.B.; Hammerschmidt, F.; Zechel, D.L.; Jia, Z.
    Crystal structure of PhnZ in complex with substrate reveals a di-iron oxygenase mechanism for catabolism of organophosphonates (2014), Proc. Natl. Acad. Sci. USA, 111, 5171-5176 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.1.3 expressed in Escherichia coli Rosetta2 cells Sinorhizobium meliloti
1.13.11.78 gene phnZ, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of wild-type and mutant C-terminally His-tagged enzymes in Escherichia coli strain DL41(DE3) uncultured bacterium HF130_AEPn_1

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.2.1.3 hanging drop vapor diffusion method, using 22%-25% (w/v) PEG3350, 0.1 M sodium cacodylate (pH 6.5), 0.2 M sodium phosphate dibasic, and 5%-10% (v/v) glycerol Sinorhizobium meliloti
1.13.11.78 purified enzyme in complex with substrate (R)-2-amino-1-hydroxyethylphosphonate or with L-tartrate, derived from the crystallization buffer, hanging drop vapour diffusion method, mixing 0.005 ml of 16 mg/ml selenomethionine-labeled protein of enzyme in complex with L-tartrate in a solution containing 5% n-octyl-beta-D-glucoside with 0.005 ml of reservoir solution containing 2.4 M ammonium sulfate and 0.15 M potassium sodium L-tartrate, 5-7 days at room temperature, the substrate-complexed enzyme is crystallized by sitting drop vapour diffusion method by mixing of 0.001 ml of protein solution with 0.001 ml of reservoir solution containing 0.1 M Bis-Tris, pH 6.5 and 20% w/v PEG 5000 monomethyl ether, X-ray diffraction structure determination and analysis at 2.1 and 1.7 A resolution, respectively, molecular replacement and modeling uncultured bacterium HF130_AEPn_1

Protein Variants

EC Number Protein Variants Comment Organism
1.2.1.3 C291A inactive Sinorhizobium meliloti
1.2.1.3 E254A inactive Sinorhizobium meliloti
1.2.1.3 E385A the mutant shows strongly reduced activity compared to the wild type enzyme Sinorhizobium meliloti
1.2.1.3 N158A the mutant shows strongly reduced activity compared to the wild type enzyme Sinorhizobium meliloti
1.2.1.3 R108A the mutant shows strongly reduced activity compared to the wild type enzyme Sinorhizobium meliloti
1.2.1.3 R290A the mutant shows strongly reduced activity compared to the wild type enzyme Sinorhizobium meliloti
1.2.1.3 R447A the mutant shows strongly reduced activity compared to the wild type enzyme Sinorhizobium meliloti
1.13.11.78 D161A site-directed mutagenesis, the mutant is inactive uncultured bacterium HF130_AEPn_1
1.13.11.78 D59A site-directed mutagenesis, the mutant is unstable uncultured bacterium HF130_AEPn_1
1.13.11.78 H104A site-directed mutagenesis, the mutant is inactive uncultured bacterium HF130_AEPn_1
1.13.11.78 H34A site-directed mutagenesis, the mutant is inactive uncultured bacterium HF130_AEPn_1
1.13.11.78 H58A site-directed mutagenesis, the mutant is inactive uncultured bacterium HF130_AEPn_1
1.13.11.78 H80A site-directed mutagenesis, the mutant shows 10fold reduced activity compared to the wild-type enzyme, reduced Fe-enzyme molar ratios exist in the variants H80A uncultured bacterium HF130_AEPn_1

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.1.3 0.0032
-
 phosphonoacetaldehyde wild type enzyme, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.0051
-
 phosphonoacetaldehyde mutant enzyme R290A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.0097
-
 phosphonoacetaldehyde mutant enzyme R108A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.018
-
 NAD+ mutant enzyme R108A, with phosphonoacetaldehyde as cosubstrate, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.019
-
 phosphonoacetaldehyde mutant enzyme E385A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.029
-
 phosphonoacetaldehyde mutant enzyme N158A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.04
-
 NAD+ mutant enzyme N158A, with phosphonoacetaldehyde as cosubstrate, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.054
-
 NAD+ mutant enzyme R447A, with phosphonoacetaldehyde as cosubstrate, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.058
-
 NAD+ wild type enzyme, with phosphonoacetaldehyde as cosubstrate, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.075
-
 NAD+ mutant enzyme R290A, with phosphonoacetaldehyde as cosubstrate, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.097
-
 glyceraldehyde 3-phosphate wild type enzyme, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.15
-
 phosphonoacetaldehyde mutant enzyme R447A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.37
-
 NAD+ mutant enzyme E385A, with phosphonoacetaldehyde as cosubstrate, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.53
-
 NAD+ wild type enzyme, with glyceraldehyde-3-phosphate as cosubstrate, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 3.3
-
 3-oxopropyl phosphonate wild type enzyme, at pH 7.5 and 30°C Sinorhizobium meliloti
1.13.11.78 additional information
-
 additional information kinetic analysis with substrate analogues, Michaelis-Menten kinetics uncultured bacterium HF130_AEPn_1
1.13.11.78 0.017
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant wild-type enzyme uncultured bacterium HF130_AEPn_1
1.13.11.78 0.022
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant Y24E uncultured bacterium HF130_AEPn_1
1.13.11.78 0.026
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant Y24F uncultured bacterium HF130_AEPn_1
1.13.11.78 0.4
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant H62A uncultured bacterium HF130_AEPn_1
1.13.11.78 0.6
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant H80A uncultured bacterium HF130_AEPn_1
1.13.11.78 1.1
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant E27A uncultured bacterium HF130_AEPn_1

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.78 Fe2+ required for catalysis, the enzyme uses a diiron oxygenase mechanism. Residue Y24 forms a transient ligand interaction at the dioxygen binding site of the two Fe2+. The first Fe ion is coordinated in a distorted octahedral geometry by Y24, H34, H58, D59, D161, and the bridging water. PhnZ ligates the second Fe2+ ion in an octahedral geometry through interactions with D59, H80, H104, and the bridging water. The second Fe is further coordinated in a bidentate fashion by the adjacent carboxylate and alpha-hydroxyl oxygens of L-tartrate uncultured bacterium HF130_AEPn_1

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.78 (2-amino-1-hydroxyethyl)phosphonate + O2 uncultured bacterium HF130_AEPn_1
-
 glycine + phosphate
-
 ?
1.13.11.78 additional information uncultured bacterium HF130_AEPn_1 the enzyme uses a di-iron oxygenase mechanism for catabolism of organophosphonates, overview ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.3 Sinorhizobium meliloti Q92UV7
-
-
1.13.11.78 uncultured bacterium HF130_AEPn_1 D0E8I5
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.1.3 Ni-NTA column chromatography and Superdex 75 gel filtration Sinorhizobium meliloti

Reaction

EC Number Reaction Comment Organism Reaction ID
1.13.11.78 (2-amino-1-hydroxyethyl)phosphonate + O2 = glycine + phosphate the enzyme uses a diiron oxygenase mechanism for catabolism of organophosphonates. The fifth histidine that is conserved in the PhnZ subclade, H62, specifically interacts with the substrate 1-hydroxy, residue Y24 forms a transient ligand interaction at the dioxygen binding site of Fe2+. Residues Y24 and E27 mediate a unique induced-fit mechanism whereby E27 specifically recognizes the 2-amino group of the bound substrate and toggles the release of Y24 from the active site, thereby creating space for molecular oxygen to bind to the second Fe2+. The 2-amino group of (R)-2 triggers an induced-fit mechanism uncultured bacterium HF130_AEPn_1
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.3 3-oxopropyl phosphonate + NAD+ + H2O
-
 Sinorhizobium meliloti ?
-
 ?
1.2.1.3 glyceraldehyde 3-phosphate + NAD+ + H2O
-
 Sinorhizobium meliloti ?
-
 ?
1.2.1.3 phosphonoacetaldehyde + NAD+ + H2O
-
 Sinorhizobium meliloti phosphonoacetate + NADH + H+
-
 ?
1.13.11.78 (2-amino-1-hydroxyethyl)phosphonate + O2
-
 uncultured bacterium HF130_AEPn_1 glycine + phosphate
-
 ?
1.13.11.78 (2-amino-1-hydroxyethyl)phosphonate + O2 enzyme PhnZ Is stereospecific for (R)-2-amino-1-hydroxyethylphosphonic acid uncultured bacterium HF130_AEPn_1 glycine + phosphate
-
 ?
1.13.11.78 additional information substrate recognition mechanism, overview uncultured bacterium HF130_AEPn_1 ?
-
 ?
1.13.11.78 additional information the enzyme uses a di-iron oxygenase mechanism for catabolism of organophosphonates, overview uncultured bacterium HF130_AEPn_1 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.2.1.3 phnY
-
 Sinorhizobium meliloti
1.13.11.78 ALOHA_HF130_AEPn_1_06c
-
 uncultured bacterium HF130_AEPn_1
1.13.11.78 phnZ
-
 uncultured bacterium HF130_AEPn_1

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.78 25
-
 assay at uncultured bacterium HF130_AEPn_1

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.1.3 0.01
-
 phosphonoacetaldehyde mutant enzyme N158A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.051
-
 phosphonoacetaldehyde mutant enzyme R108A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.076
-
 phosphonoacetaldehyde mutant enzyme R447A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.098
-
 glyceraldehyde 3-phosphate wild type enzyme, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.12
-
 phosphonoacetaldehyde mutant enzyme R290A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 0.19
-
 phosphonoacetaldehyde mutant enzyme E385A, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 1.5
-
 3-oxopropyl phosphonate wild type enzyme, at pH 7.5 and 30°C Sinorhizobium meliloti
1.2.1.3 2.2
-
 phosphonoacetaldehyde wild type enzyme, at pH 7.5 and 30°C Sinorhizobium meliloti
1.13.11.78 2
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant H62A uncultured bacterium HF130_AEPn_1
1.13.11.78 3
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant E27A uncultured bacterium HF130_AEPn_1
1.13.11.78 4
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant H80A uncultured bacterium HF130_AEPn_1
1.13.11.78 8
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant Y24E uncultured bacterium HF130_AEPn_1
1.13.11.78 11
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant wild-type enzyme uncultured bacterium HF130_AEPn_1
1.13.11.78 11
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant Y24F uncultured bacterium HF130_AEPn_1

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.78 6.8 7 assay at uncultured bacterium HF130_AEPn_1

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.3 NAD+
-
 Sinorhizobium meliloti

General Information

EC Number General Information Comment Organism
1.13.11.78 evolution enzyme PhnZ belongs to a large family of hydrolytic enzymes, the HD-phosphohydrolase superfamily, which comprises enzymes that use a strictly conserved His-Asp sequence motif to bind active site metal ions. Structural comparisons of PhnZ reveal an evolutionary connection between Fe(II)-dependent hydrolysis of phosphate esters and oxidative carbon-phosphorus or carbon-carbon bond cleavage, thus uniting the diverse chemistries that are found in the HD superfamily. PhnZ is a structural homologue of myo-inositol oxygenase and Fe(II)-dependent phosphohydrolases uncultured bacterium HF130_AEPn_1
1.13.11.78 additional information enzyme PhnZ has an active site containing two Fe ions coordinated by four histidines and two aspartates that is strikingly similar to the carbon-carbon bond cleaving enzyme, myo-inositol-oxygenase. The exception is residue Y24, which forms a transient ligand interaction at the dioxygen binding site of the second Fe2+. Structure comparisons and substrate binding structures, active site structure, detailed overview uncultured bacterium HF130_AEPn_1
1.13.11.78 physiological function the enzymes PhnY and PhnZ comprise an oxidative catabolic pathway that enables marine bacteria to use 2-aminoethylphosphonic acid as a source of inorganic phosphate. PhnZ is notable for catalyzing the oxidative cleavage of a carbon-phosphorus bond using Fe(II) and dioxygen uncultured bacterium HF130_AEPn_1

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.13.11.78 2.72
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant E27A uncultured bacterium HF130_AEPn_1
1.13.11.78 5
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant H62A uncultured bacterium HF130_AEPn_1
1.13.11.78 6.67
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant H80A uncultured bacterium HF130_AEPn_1
1.13.11.78 347.6
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant wild-type enzyme uncultured bacterium HF130_AEPn_1
1.13.11.78 363.6
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant Y24E uncultured bacterium HF130_AEPn_1
1.13.11.78 423.1
-
 (2-amino-1-hydroxyethyl)phosphonate pH 7.0, 25°C, recombinant mutant Y24F uncultured bacterium HF130_AEPn_1