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Literature summary extracted from

  • Frank, A.; Siirola, E.; Kroutil, W.; Grogan, G.
    Mutational analysis of the C-C bond cleaving enzyme phloretin hydrolase from Eubacterium ramulus (2014), Topics Catal., 57, 376-384.
No PubMed abstract available

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.7.1.4 gene phy, DNA and amino acid sequence determination and analysis, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Eubacterium ramulus
3.7.1.24 gene phlg, sequence comparison Pseudomonas fluorescens

Protein Variants

EC Number Protein Variants Comment Organism
3.7.1.4 A126S site-directed mutagenesis Eubacterium ramulus
3.7.1.4 F218Y site-directed mutagenesis Eubacterium ramulus
3.7.1.4 additional information mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) results in variants that were either poorly expressed, or of much reduced activity. Mutation of Tyr115 and His203, thought to bind the phenol groups in the 1-and 3-positions of the phloroglucinol ring respectively, results in variants of 15-fold reduced activity and an inactive variant Eubacterium ramulus
3.7.1.24 E160A site-directed mutagenesis, almost inactive enzyme Pseudomonas fluorescens
3.7.1.24 E274A site-directed mutagenesis, a mutant of very low activity Pseudomonas fluorescens
3.7.1.24 H214A site-directed mutagenesis, the mutant shows an unaltered KM but reduced catalytic efficiency with diacetyl phloroglucinol compared to the wild-type Pseudomonas fluorescens
3.7.1.24 H270A site-directed mutagenesis, inactive enzyme Pseudomonas fluorescens
3.7.1.24 additional information mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) results in variants that are either poorly expressed, or of much reduced activity. Mutation of Tyr115 and His203, thought to bind the phenol groups in the 1-and 3-positions of the phloroglucinol ring respectively, results in variants of 15fold reduced activity and an inactive variant Pseudomonas fluorescens
3.7.1.24 Y121A site-directed mutagenesis, the mutant shows reduced KM and catalytic efficiency with diacetyl phloroglucinol compared to the wild-type Pseudomonas fluorescens

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.7.1.4 Zn2+ a Zn-dependent C-C hydrolase, His123, His251, Glu154 and Glu255 are conserved zinc binding residues Eubacterium ramulus
3.7.1.24 Zn2+ a Zn-dependent C-C hydrolase, bound in the active site, chelated by four amino acid side-chains, His270, His129, Glu274 and Glu160, might also have structural role in Phlg Pseudomonas fluorescens

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.7.1.4 30000
-
 2 * 30000, recombinant enzyme, SDS-PAGE Eubacterium ramulus
3.7.1.4 55000
-
 recombinant enzyme, gel filtration Eubacterium ramulus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.7.1.4 phloretin + H2O Eubacterium ramulus
-
 phloretate + phloroglucinol
-
 ?
3.7.1.24 2,4-diacetylphloroglucinol + H2O Pseudomonas fluorescens
-
 2-acetylphloroglucinol + acetate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.7.1.4 Eubacterium ramulus Q715L4 Eubacterium ramulus is part of the natural gut flora in humans, gene phy
-
3.7.1.24 Pseudomonas fluorescens Q4K423
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.7.1.4 recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3) Eubacterium ramulus

Reaction

EC Number Reaction Comment Organism Reaction ID
3.7.1.24 2,4-diacetylphloroglucinol + H2O = 2-acetylphloroglucinol + acetate catalytic reaction mechanism, overview. A water molecule, bound by the zinc, is responsible for initiating the C–C bond cleavage reaction through nucleophilic attack at the acyl carbonyl carbon atom Pseudomonas fluorescens
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.7.1.4 additional information no activity with 1,3-diphenyl 1,3-propanedione and diacetylphloroglucinol, a substrate of diacetyl phloroglucinol hydrolase, Phlg Eubacterium ramulus ?
-
 ?
3.7.1.4 phloretin + H2O
-
 Eubacterium ramulus phloretate + phloroglucinol
-
 ?
3.7.1.4 phloretin + H2O phloretin hydrolase catalyzes the hydrolysis of the dihydrochalcone phloretin to phloroglucinol and phloretic acid, performing a formal retro-Friedel-Crafts acylation reaction on its substrate, it is a C-C bond cleaving enzyme Eubacterium ramulus phloretate + phloroglucinol
-
 ?
3.7.1.24 2,4-diacetylphloroglucinol + H2O
-
 Pseudomonas fluorescens 2-acetylphloroglucinol + acetate
-
 ?
3.7.1.24 2,4-diacetylphloroglucinol + H2O modeling of a theoretical transition state for the substrate hydrolysis into the active site revealing that a water molecule, bound by the zinc, can be responsible for initiating the C-C bond cleavage reaction through nucleophilic attack at the acyl carbonyl carbon atom. Residue Asn132 in Phlg is thought to make a hydrogen bond to the acyl group of the native substrate in the enzyme. Tyr121 is thought to hydrogen bond to the phenol group in the 1-position of the aromatic ring between the two acetyl groups in the substrate Pseudomonas fluorescens 2-acetylphloroglucinol + acetate
-
 ?

Subunits

EC Number Subunits Comment Organism
3.7.1.4 homodimer 2 * 30000, recombinant enzyme, SDS-PAGE Eubacterium ramulus

Synonyms

EC Number Synonyms Comment Organism
3.7.1.4 Phy
-
 Eubacterium ramulus
3.7.1.24 diacetyl phloroglucinol hydrolase
-
 Pseudomonas fluorescens
3.7.1.24 diacetylphloroglucinol hydrolase
-
 Pseudomonas fluorescens
3.7.1.24 Phlg
-
 Pseudomonas fluorescens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.7.1.4 30
-
 assay at Eubacterium ramulus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.7.1.4 7.5
-
 assay at Eubacterium ramulus

General Information

EC Number General Information Comment Organism
3.7.1.4 additional information comparison of the enzyme phloretin hydrolase Phy from Eubacterium ramulus with the diacetyl phloroglucinol hydrolase (Phlg) from Pseudomonas fluorescens, which catalyses a similar, hydrolytic, de-acylation of its substrate, homology modeling of Phy based on the structure of Phlg, PDB ID 3HWP Eubacterium ramulus
3.7.1.4 physiological function the enzyme catalyses the degradation of plant-derived dihydrochalcone phloretin Eubacterium ramulus
3.7.1.24 evolution the C-C hydrolase enzyme exhibits a Bet v1-like fold rather than the alpha/beta hydrolase fold common to C-C hydrolases Pseudomonas fluorescens
3.7.1.24 malfunction mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) results in variants that are either poorly expressed, or of much reduced activity. Mutation of Tyr115 and His203, thought to bind the phenol groups in the 1-and 3-positions of the phloroglucinol ring respectively, results in variants of 15fold reduced activity and an inactive variant Pseudomonas fluorescens
3.7.1.24 additional information the enzyme structure, PDB ID 3HWP, is used for a homology model of phloretin hydrolase, Phy, EC 3.7.1.4, from Eubacterium ramulus. Active site structure of the enzyme Phlg, overview Pseudomonas fluorescens