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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.

Phlorizin ( 1 ) was anaerobically incubated with human intestinal bacteria and five biotransformation products ( 2 – 6 ) were obtained. Their structures were elucidated as phloretin ( 2 ), phloroglucinol ( 3 ), phloretic acid ( 4 ), 2,3,4-trihydroxybenzenepropanoic acid ( 5 ), and phloretic acid methyl ester ( 6 ) on the basis of their spectroscopic data. Using high-performance liquid chromatography equipped with a diode array detector, chromatographic separation of 1 – 4 was performed on an analytical C 18 column. The time course of the biotransformation was studied to probe into the biotransformation mechanism of 1 by human intestinal flora. The abilities of isolated strains to transform 1 were also investigated in order to find an optimal transformation strain. In addition, the inhibitory activity of parent compound 1 and its three main biotransformation products 2 – 4 on mushroom tyrosinase was estimated. The result showed that 2 has better inhibitory effect on tyrosinase activity than 1 .

Micropropagated shoots of wild cherry (Prunus avium L.) produced roots in auxin-free medium. Phloroglucinol (PG) increased the proportion of shoots that rooted, while phloretic acid reduced this response in medium with or without PG, and cancelled the promotive effect of PG. Concentration of PG also significantly affected rooting in media with and without auxin. The proportion of shoots rooting in media containing auxin, or auxin plus PG, increased with the number of successive subculture, but the proportion that rooted with PG alone was unaffected by the number of subcultures. Before the shoots had become responsive to auxin, 1 mM PG was more effective than auxin in inducing root formation.

SummaryTwo phloridzin breakdown products, phloroglucinol and phloretic acid, more than doubled the proportion of M.7 apple rootstock shoots which rooted when cultured in vitro with auxins. Caffeic acid, catechol and pyrogallol did not significantly increase rooting.

Summary A number of factors that affect the in vitro rooting of M.9 both during the shoot proliferation and the root-inducing phase of the micropropagation process have been examined. Work with the phenolic compound phloroglucinol (1, 3, 5 trihydroxybenzene) has shown that it can precondition shoots to root whilst shoots are multiplying on a cytokinin-based medium. Excision of single shoots and exposure to auxin and phloroglucinol (PG) also stimulated rooting compared to auxin-alone controls, although the size of the effect was not as great as that recorded during shoot multiplication. The easier-to-root M.26 was not affected by PG at either stage of the micropropagation process. Ageing of shoot cultures increased the capacity for auxin-induced rooting but shoot etiolation was without effect. A number of phenolic compounds at 10 -3 M, were assessed as IAA synergists during the root- initiation phase. Of the simple phenols only PG and hydroquinone had any effect on root number. Salicylic acid completely inhibited the root-inducing effect of IAA. The phenylpropanoids ferulic acid and umbelliferone significantly reduced rooting whilst chlorogenic acid increased root number. Phloridzin and its degradation products phloretin, phloretic acid and PG all increased root number compared to IAA controls.

The activity of an enzyme system decomposing phloridzin was investigated in fruitbearing and nonbearing spurs of apple trees, Landsberger Reinette cv., throughout vegetation. Acetone powder obtained from xylem sap of apple spurs was incubated with phloridzinsubstratum in citric buffer at pH 5.5 for 12, 18 and 24 h at 30 °C. A paper and thin-layer chromatography as well aa a spectrophotometric assay were employed for tentative identification of enzymic degradation products. Phloretic acid (PA), co-factor of IAA-oxidase, as well as phloretin (Pin), and phloroglucinol (PI) were found after the digestion of phloridzin. The chromatographed enzyme reaction products were measured densitometrically. The activity of the enzyme system was estimated by its efficiency in PA production and phloridzin disappearance. Obtained values, expressed in percentages, showed that the enzyme activity in fruitbearing spurs was much higher than in nonbearing ones; 30 and 10% of released PA in July, respectively. Because fruitbearing spurs of the apple tree are possibly additionally supplied with auxin translocated from developing seeds, an adaptive character of the enzyme system producing PA, a known auxin repressor, is suggested.

Phloretin, a 7,8-dihydrochalcone of plant origin, and the high molecular weight (less than 15,000) polyphloretinphosphate (PPP) polymers are potent inhibitors of iodothyronine monodeiodinase activity from rat liver microsomal preparations, whereas phlorizin, the 2'-O-glucoside of phloretin, is inactive. The polymers, differing in degree of phosphorylation-dependent polymerization, exhibited a concentration-dependent, and ultimately complete, inhibition of deiodinase activity with an IC50 between 0.2 and 0.5 micrograms PPP/ml. Phloretin inhibition, on the other hand, was cofactor (DTE) competitive, with a Ki = 0.75 microM. 2',4',6',3,4- Pentahydroxychalcone, which has a substitution pattern in the A-ring identical to that of phloretin, was the only active inhibitor (IC50 = 8 microM) among several derivatives tested. The phloretin biodegradation products, phloretic acid and phloroglucinol, and its biosynthetic precursors, monomeric cinnamic acid and cinnamic acid derivatives, were inactive in concentrations up to 100 microM. The X-ray crystal structure analysis of phlorizin dihydrate showed that the molecule is planar and fully extended, similar to the conformation observed in chalcone structures that are characterized by an alpha, beta-unsaturated bond between phenol rings. Comparison of the planar phlorizin crystal structure with a skewed or antiskewed thyroid hormone conformation revealed that the beta-D-glucose moiety does not share any of the thyroid hormone's conformational space, and that the best structural homology is found with the antiskewed conformation of 3',5',3-triiodothyronine, the natural deiodinase substrate that also inhibits further deiodination.

A variety of fungi were shown to degrade phloridzin to phloretin, phloroglucinol and phloretic acid. Phloretic acid in turn was metabolized to p -hydroxybenzoic acid. Aspergillus niger yielded cell-free preparations which catalyzed the hydrolysis of phloretin to phloroglucinol and phloretic acid.

The metabolism of phloretin (2',4',6',4-tetrahydroxydihydrochalcone) was studied in rats. Approx. half of the intragastric dose (0.75 mmol/kg) was excreted in the urine, mainly within two days. Small initial amounts of phloretin were found, however most of the metabolites were degradation products. The latter included phloroglucinol and, in larger amounts, phloretic acid and related metabolites formed by its dehydrogenation, beta-oxidation and glycine conjugation. Phloroglucinol, administered in similar experiments, was rapidly (90% within 24 h) excreted in the urine, either unchanged or as conjugates (glucuronide/sulphate). Incubation of phloretin and its glucoside phloridzin with rat-caecal micro-organisms resulted in the formation of phloroglucinol and phloretic acid. The degradative pathways of metabolism of dihydrochalcones and other flavonoids are discussed.

Erwinia herbicola Y46 degrades phloridzin to yield phloretin, phloroglucinol, and phloretic acid, when grown on defined medium containing phloridzin as the sole source of carbon. The identities of the intermediates isolated from culture filtrates were established by co-chromatography and by ultraviolet absorption spectra. Only 3 of 11 strains of this species, and none of the 12 species of bacterial phytopathogens tested could effect this breakdown. Some of the latter organisms possessed β-glucosidase activity which liberated d -glucose from phloridzin. The enzyme phloretin hydrolase was purified from cells of E. herbicola Y46 grown on Yeast Beef Broth, by treatment of crude extracts with protamine sulfate, ammonium sulfate precipitation, elution from calcium phosphate gel, elution from diethylaminoethyl-cellulose, and concentration by ultrafiltration. The final preparation was free of β-glucosidase, had a specific activity of 213 units per mg of protein, and represented a 142-fold purification over the crude extract. The enzyme had a p H optimum of 6.7 to 6.8, and produced only phloroglucinol and phloretic acid as products of phloretin breakdown, there being an equimolar relationship between the cleavage of phloretin and the formation of the products. The Michaelis constant (K m ) for the enzyme with phloretin as substrate was 3.8 × 10 −5 m , and the enzyme was sensitive to Hg 2+ and Cu 2+ ions. Phloroglucinol, phloretic acid, p -chloromercuribenzoate and iodoacetamide were without effect on the activity. The enzyme did not react with phloridzin, naringin, or naringenin. The physiological significance of the results is discussed.

A modification of the vanillin-HCl reagent has been applied to the assay of phloroglucinol in the presence of phloretin. The interference of phloretin in the assay was reduced to acceptable levels, and the modified reagents permitted the satisfactory assay of the phloretin hydrolase from Erwinia herbicola , which was not possible with the original reagent of Zaprometov (1). In the modified method, 0–25 μg of phloroglucinol in water was treated with 4.5 ml concentrated HCl, followed by 0.5 ml of 1.0% vanillin in absolute ethanol. The absorbance of the mixture was read at 480 mμ any time between 2 and 20 min after mixing. Phloretin gave a low response to the reagent. Phloretic acid, phloridzin, and catechol did not react, whereas resorcinol strongly reacted with both the original and modified reagents.

PHENOLIC compounds and their glycosides are widespread in plants. They have uncertain metabolic roles, although they are regarded as metabolic by-products conferring resistance to pathogens1,2. Phloridzin, the 2′-glucoside of phloretic acid, for example, is unique to species of Malus and is the major phenolic compound of commercial apple trees (Malus pumila Mill), representing 3–7% of the dry weight of leaves as well as occurring in bark and roots3. It is not involved in resistance to apple scab as formerly supposed4, but affects the metabolism of many organisms. It inhibits growth of wheat coleoptiles5 and tomato roots6 but more than doubles photosynthetic rates in Elodea7,8. In animals it inhibits the utilisation of carbohydrates9, and one of the degradation products4,10 of phloridzin, phloroglucinol, acts synergistically with auxin to promote growth of oat mesocotyls11. There have been indications that phloridzin, and phloroglucinol, promote the expansion of apple leaves12 and the rooting of apple shoots13, respectively. This suggests that phloridzin may have important effects on Malus. I have now shown that phloridzin promotes the growth of cultured apple shoots. The effect is pronounced and has practical application.