Literature Information

Title	Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme
Author	Lilian Schoefer, Annett Braune, Michael Blaut
DOI	10.1128/AEM.70.10.6131-6137.2004
Abstract	Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37°C and 7.0, respectively. The Km for phloretin was 13 ± 3 μM and the kcat was 10 ± 2 s−1. The enzyme did not transform phloretin-2′-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl2 to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.

Experimental results

• Enzyme

Uniprot ID: Q715L4

Protein: Phloretin hydrolase

Organism: Eubacterium ramulus

Length: 274 AA

Taxonomic identifier: 39490 [NCBI]

• Pfam

Source	Domain	Start	End	E-value (Domain)	Coverage
Pfam-A	DAPG_hydrolase	57	268	2.1e-65	0.978

Program: hmmscan

Version: 3.1b2 (February 2015)

Method: hmmscan --domtblout hmmscan.tbl --noali -E 1e-5 pfam query.fa

Date: Mon Jul 20 14:32:16 2020

Description:

DAPG_hydrolase

Pfam

This domain is found in 2,4-diacetylphloroglucinol hydrolase PhiG present in Pseudomonas fluorescens. 2,4-diacetylphloroglucinol hydrolase is the gene product of PhiG that is responsible for cleaving toxic 2,4-diacetylphloroglucinol (DAPG). The small N-terminal region of the domain is involved in dimerization through hydrogen bonding of the dimer interface. The C-terminal catalytic region resembles the tetracenomycin aromatase/cyclase and has a Bet v1-like fold. DAPG PhiG is the first discovered hydrolase whose catalytic domain belongs to the Bet v1-like fold, rather than the classical alpha/beta-fold hydrolases¹.

InterPro

This domain is found in 2,4-diacetylphloroglucinol hydrolase PhiG present in Pseudomonas fluorescens. 2,4-diacetylphloroglucinol hydrolase is the gene product of PhiG that is responsible for cleaving toxic 2,4-diacetylphloroglucinol (DAPG). The small N-terminal region of the domain is involved in dimerization through hydrogen bonding of the dimer interface. The C-terminal catalytic region resembles the tetracenomycin aromatase/cyclase and has a Bet v1-like fold. DAPG PhiG is the first discovered hydrolase whose catalytic domain belongs to the Bet v1-like fold, rather than the classical alpha/beta-fold hydrolases¹.

Information is taken from Pfam and InterPro web site.

• Reaction

phloretin + H₂O ⇒ Phloretic acid + phloroglucinol

References