Literature summary for 3.7.1.24 extracted from

He, Y.-X.; Huang, l.; Xue, Y.; Fei, X.; Teng, Y.-B.; Rubin-Pitel, S.B.; Zhao, h.; Zhou C.-Z.
Crystal structure and computational analyses provide insights into the catalytic mechanism of 2,4-diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens (2010), J. Biol. Chem., 285, 4603-4611.

Cloned(Commentary)

Cloned (Comment)	Organism
gene phlG, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) or B834 (DE3), the latter for the selenomethionine-labeled enzyme	Pseudomonas fluorescens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified wild-type and selenomethionine-labeled enzymes, hanging drop vapor diffusion technique, mixing of 0.001 ml of 20 mg/ml protein sample with an equal volume of the reservoir solution containing 20% PEG 4000, 17% isopropyl alcohol, and 0.1 M sodium citrate, pH 5.6, crystals appear in 1-2 days and reach maximum size in 1 week, at 16°C, X-ray diffraction structure determination and analysis at 2.0 A resolution, MAD phasing method, modeling	Pseudomonas fluorescens

Crystallization (Comment)

Organism

purified wild-type and selenomethionine-labeled enzymes, hanging drop vapor diffusion technique, mixing of 0.001 ml of 20 mg/ml protein sample with an equal volume of the reservoir solution containing 20% PEG 4000, 17% isopropyl alcohol, and 0.1 M sodium citrate, pH 5.6, crystals appear in 1-2 days and reach maximum size in 1 week, at 16°C, X-ray diffraction structure determination and analysis at 2.0 A resolution, MAD phasing method, modeling

Pseudomonas fluorescens

Protein Variants

Protein Variants	Comment	Organism
E160A	site-directed mutagenesis, the mutation of the zinc binding residue leads to reduced activity compared to the wild-type enzyme	Pseudomonas fluorescens
E274A	site-directed mutagenesis, the mutation of the zinc binding residue leads to reduced activity compared to the wild-type enzyme	Pseudomonas fluorescens
H214A	site-directed mutagenesis, mutation of a polar residue that is predicted to form hydrogen bond with the polar groups of the substrate, the mutant shows reduced activity compared to the wild-type enzyme	Pseudomonas fluorescens
H214Q	site-directed mutagenesis, mutation of a polar residue that is predicted to form hydrogen bond with the polar groups of the substrate, the mutant shows reduced activity compared to the wild-type enzyme	Pseudomonas fluorescens
H270A	site-directed mutagenesis, inactive mutant	Pseudomonas fluorescens
N132A	site-directed mutagenesis, mutation of a polar residue that is predicted to form hydrogen bond with the polar groups of the substrate, the mutant shows reduced activity compared to the wild-type enzyme	Pseudomonas fluorescens
Y121A	site-directed mutagenesis, mutation of a polar residue that is predicted to form hydrogen bond with the polar groups of the substrate, the mutant shows reduced activity compared to the wild-type enzyme	Pseudomonas fluorescens
Y229A	site-directed mutagenesis, mutation of a polar residue that is predicted to form hydrogen bond with the polar groups of the substrate, the mutant shows reduced activity compared to the wild-type enzyme	Pseudomonas fluorescens
Y229F	site-directed mutagenesis, mutation of a polar residue that is predicted to form hydrogen bond with the polar groups of the substrate, the mutant shows reduced activity compared to the wild-type enzyme	Pseudomonas fluorescens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.041	-	2,4-diacetylphloroglucinol	recombinant mutant E160A, pH 7.1, 25°C	Pseudomonas fluorescens
0.042	-	2,4-diacetylphloroglucinol	recombinant mutant E274A, pH 7.1, 25°C	Pseudomonas fluorescens
0.18	-	2,4-diacetylphloroglucinol	recombinant mutant H214Q, pH 7.1, 25°C	Pseudomonas fluorescens
0.242	-	2,4-diacetylphloroglucinol	recombinant mutant Y121A, pH 7.1, 25°C	Pseudomonas fluorescens
0.268	-	2,4-diacetylphloroglucinol	recombinant mutant Y229F, pH 7.1, 25°C	Pseudomonas fluorescens
0.272	-	2,4-diacetylphloroglucinol	recombinant wild-type enzyme, pH 7.1, 25°C	Pseudomonas fluorescens
0.32	-	2,4-diacetylphloroglucinol	recombinant mutant H214A, pH 7.1, 25°C	Pseudomonas fluorescens
0.61	-	2,4-diacetylphloroglucinol	recombinant mutant N132A, pH 7.1, 25°C	Pseudomonas fluorescens
0.873	-	2,4-diacetylphloroglucinol	recombinant mutant Y229A, pH 7.1, 25°C	Pseudomonas fluorescens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Zn2+	a Zn-dependent C-C hydrolase, the metal ion may play an essential role in catalysis and is identified as bound inside the hydrophobic/amphiphilic pocket, coordinated by His129(beta5), Glu160(beta6), His270(alpha10), Glu274(alpha10), and a water molecule with pentagonal bipyramidal coordination geometry. It may play an important structural role in stabilizing the PhlG catalytic domain by holding the strands beta5 and beta6 and the helix alpha10 together	Pseudomonas fluorescens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2,4-diacetylphloroglucinol + H2O	Pseudomonas fluorescens	-	2-acetylphloroglucinol + acetate	-	?
2,4-diacetylphloroglucinol + H2O	Pseudomonas fluorescens Pf-5	-	2-acetylphloroglucinol + acetate	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas fluorescens	Q4K423	gene phlG	-
Pseudomonas fluorescens Pf-5	Q4K423	gene phlG	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli	Pseudomonas fluorescens

Reaction

Reaction	Comment	Organism	Reaction ID
2,4-diacetylphloroglucinol + H2O = 2-acetylphloroglucinol + acetate	cleavage of the 2,4-diacetylphloroglucinol C-C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. Residues Tyr121, Tyr229, and Asn132, which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation, catalytic reaction mechanism, overview	Pseudomonas fluorescens	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2,4-diacetylphloroglucinol + H2O	-	Pseudomonas fluorescens	2-acetylphloroglucinol + acetate	-	?
2,4-diacetylphloroglucinol + H2O	2,4-diacetylphloroglucinol hydrolase catalyzes hydrolytic C-C bond cleavage, cleaving one of the C-C bonds linking the acetyl groups to the phenolic ring, of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry, strict substrate specificity, no degradation of other compounds of similar structure such as monoacetylphloroglucinol and triacetylphloroglucinol	Pseudomonas fluorescens	2-acetylphloroglucinol + acetate	-	?
2,4-diacetylphloroglucinol + H2O	-	Pseudomonas fluorescens Pf-5	2-acetylphloroglucinol + acetate	-	?
2,4-diacetylphloroglucinol + H2O	2,4-diacetylphloroglucinol hydrolase catalyzes hydrolytic C-C bond cleavage, cleaving one of the C-C bonds linking the acetyl groups to the phenolic ring, of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry, strict substrate specificity, no degradation of other compounds of similar structure such as monoacetylphloroglucinol and triacetylphloroglucinol	Pseudomonas fluorescens Pf-5	2-acetylphloroglucinol + acetate	-	?

Subunits

Subunits	Comment	Organism
dimer	overall structure of PhlG dimer, modeling, overview	Pseudomonas fluorescens

Synonyms

Synonyms	Comment	Organism
Phlg	-	Pseudomonas fluorescens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Pseudomonas fluorescens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.000026	-	2,4-diacetylphloroglucinol	recombinant mutant E274A, pH 7.1, 25°C	Pseudomonas fluorescens
0.000081	-	2,4-diacetylphloroglucinol	recombinant mutant E160A, pH 7.1, 25°C	Pseudomonas fluorescens
0.05	-	2,4-diacetylphloroglucinol	recombinant mutant Y121A, pH 7.1, 25°C	Pseudomonas fluorescens
0.21	-	2,4-diacetylphloroglucinol	recombinant mutant Y229A, pH 7.1, 25°C	Pseudomonas fluorescens
0.58	-	2,4-diacetylphloroglucinol	recombinant mutant H214Q, pH 7.1, 25°C	Pseudomonas fluorescens
0.74	-	2,4-diacetylphloroglucinol	recombinant mutant N132A, pH 7.1, 25°C	Pseudomonas fluorescens
1.4	-	2,4-diacetylphloroglucinol	recombinant mutants Y229F and H214A, pH 7.1, 25°C	Pseudomonas fluorescens
4.7	-	2,4-diacetylphloroglucinol	recombinant wild-type enzyme, pH 7.1, 25°C	Pseudomonas fluorescens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.1	-	assay at	Pseudomonas fluorescens

General Information

General Information	Comment	Organism
evolution	the amino acid sequence of PhlG displays 25-37% sequence identity to several hypothetical proteins and the phloretin hydrolase (EC 3.7.1.4) from Eubacterium ramulus. Neither PhlG nor phloretin hydrolase possess sequence homology to other C-C bond-cleaving hydrolases or motifs typical of the alpha/beta hydrolase family, suggesting these two enzymes belong to a distinct hydrolase family, the enzyme structure with a Bet v1-like fold also distinguishes PhlG from the classical alpha/beta-fold hydrolases. PhlG is a hydrolase whose catalytic domain belongs to the Bet v1-like fold and belongs to another family within the Bet v1-like superfamily. The most important characteristic of Bet v1-like proteins is the presence of an interior hydrophobic/amphiphilic pocket accessible to the exterior, present in the C-terminal domain of PhlG	Pseudomonas fluorescens
additional information	three-dimensional structure analysis: the overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes enzyme PhlG from the classical alpha/beta-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site, computational docking studies	Pseudomonas fluorescens

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.00062	-	2,4-diacetylphloroglucinol	recombinant mutant E274A, pH 7.1, 25°C	Pseudomonas fluorescens
0.002	-	2,4-diacetylphloroglucinol	recombinant mutant E160A, pH 7.1, 25°C	Pseudomonas fluorescens
0.21	-	2,4-diacetylphloroglucinol	recombinant mutant Y121A, pH 7.1, 25°C	Pseudomonas fluorescens
1.1	-	2,4-diacetylphloroglucinol	recombinant mutant Y229A, pH 7.1, 25°C	Pseudomonas fluorescens
1.2	-	2,4-diacetylphloroglucinol	recombinant mutant N132A, pH 7.1, 25°C	Pseudomonas fluorescens
3.3	-	2,4-diacetylphloroglucinol	recombinant mutant H214Q, pH 7.1, 25°C	Pseudomonas fluorescens
4.3	-	2,4-diacetylphloroglucinol	recombinant mutant H214A, pH 7.1, 25°C	Pseudomonas fluorescens
5.2	-	2,4-diacetylphloroglucinol	recombinant mutant Y229F, pH 7.1, 25°C	Pseudomonas fluorescens
17	-	2,4-diacetylphloroglucinol	recombinant wild-type enzyme, pH 7.1, 25°C	Pseudomonas fluorescens