Topics in Catalysis

, Volume 57, Issue 5, pp 376–384 | Cite as

Mutational Analysis of the C–C Bond Cleaving Enzyme Phloretin Hydrolase from Eubacterium ramulus

  • Annika Frank
  • Elina Siirola
  • Wolfgang Kroutil
  • Gideon GroganEmail author
Original Paper


Phloretin hydrolase from Eubacterium ramulus (Phy) catalyzes the hydrolysis of the dihydrochalcone phloretin to phloroglucinol and phloretic acid, performing a formal retro- Friedel–Crafts acylation reaction on its substrate. Its closest sequence homolog, of 25 % amino acid sequence identity, is diacetyl phloroglucinol hydrolase (Phlg) from Pseudomonas fluorescens, which catalyses a similar, hydrolytic, de-acylation of its substrate. The structure of Phlg has been determined and a catalytic mechanism proposed (J Biol Chem 285:4603–4611, 2010). In order to compare the catalytic characteristics of Phy with Phlg, the gene encoding Phy was expressed and the enzyme purified and crystallised. An X-ray fluorescence scan identified zinc within the crystals. A homology model of Phy, based on the structure of Phlg (PDB code 3HWP), informed the construction of a point mutant library of the enzyme, targeting residues shared with Phlg that are thought to be involved in zinc binding and the recognition of acyl and phenol functionality on the aromatic ring of the substrates. Mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) resulted 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, resulted in variants of 15-fold reduced activity and an inactive variant. These results are suggestive of conservation of some aspects of mechanism and substrate recognition between Phy and Phlg, and of the catalytic characteristics of Zn-dependent C–C hydrolases of this type in general.


C–C bond hydrolase Metalloenzyme Phloretin Phloretic acid Dihydrochalcone 



This research was supported by Marie Curie Network for Initial Training fellowships to A.F. and E.S. in the project BIOTRAINS (FP7-PEOPLE-ITN-2008-238531). This work was carried out with the support of the Diamond Light Source synchrotron in Oxford, United Kingdom.


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Annika Frank
    • 1
  • Elina Siirola
    • 2
  • Wolfgang Kroutil
    • 2
  • Gideon Grogan
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of YorkYorkUK
  2. 2.Department of Chemistry, Organic and Bioorganic ChemistryUniversity of GrazGrazAustria

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