Literature summary for 1.13.11.78 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 .

Search for more literature for 1.13.11.78

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
D161A	site-directed mutagenesis, the mutant is inactive	uncultured bacterium HF130_AEPn_1
D59A	site-directed mutagenesis, the mutant is unstable	uncultured bacterium HF130_AEPn_1
H104A	site-directed mutagenesis, the mutant is inactive	uncultured bacterium HF130_AEPn_1
H34A	site-directed mutagenesis, the mutant is inactive	uncultured bacterium HF130_AEPn_1
H58A	site-directed mutagenesis, the mutant is inactive	uncultured bacterium HF130_AEPn_1
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]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	kinetic analysis with substrate analogues, Michaelis-Menten kinetics	uncultured bacterium HF130_AEPn_1
0.017	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant wild-type enzyme	uncultured bacterium HF130_AEPn_1
0.022	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant Y24E	uncultured bacterium HF130_AEPn_1
0.026	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant Y24F	uncultured bacterium HF130_AEPn_1
0.4	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant H62A	uncultured bacterium HF130_AEPn_1
0.6	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant H80A	uncultured bacterium HF130_AEPn_1
1.1	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant E27A	uncultured bacterium HF130_AEPn_1

Metals/Ions

Metals/Ions	Comment	Organism	Structure
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)

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

Organism

Organism	UniProt	Comment	Textmining
uncultured bacterium HF130_AEPn_1	D0E8I5	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
(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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(2-amino-1-hydroxyethyl)phosphonate + O2	-	uncultured bacterium HF130_AEPn_1	glycine + phosphate	-	?
(2-amino-1-hydroxyethyl)phosphonate + O2	enzyme PhnZ Is stereospecific for (R)-2-amino-1-hydroxyethylphosphonic acid	uncultured bacterium HF130_AEPn_1	glycine + phosphate	-	?
additional information	substrate recognition mechanism, overview	uncultured bacterium HF130_AEPn_1	?	-	?
additional information	the enzyme uses a di-iron oxygenase mechanism for catabolism of organophosphonates, overview	uncultured bacterium HF130_AEPn_1	?	-	?

Synonyms

Synonyms	Comment	Organism
ALOHA_HF130_AEPn_1_06c	-	uncultured bacterium HF130_AEPn_1
phnZ	-	uncultured bacterium HF130_AEPn_1

Temperature Optimum [°C]

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

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
2	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant H62A	uncultured bacterium HF130_AEPn_1
3	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant E27A	uncultured bacterium HF130_AEPn_1
4	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant H80A	uncultured bacterium HF130_AEPn_1
8	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant Y24E	uncultured bacterium HF130_AEPn_1
11	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant wild-type enzyme	uncultured bacterium HF130_AEPn_1
11	-	(2-amino-1-hydroxyethyl)phosphonate	pH 7.0, 25°C, recombinant mutant Y24F	uncultured bacterium HF130_AEPn_1

pH Optimum

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

General Information

General Information	Comment	Organism
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
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
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]

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