Metabolism of phenolic amines : degradation ofdl-synephrine by Arthrobacter synerphrium sp.Nov. Ph.D

Abstract

Among the natural phenolic amines, those derived from phenylalanine form an important group and are sometimes distinguished as protoalkaloids. Although the role of phenolic amines in animal metabolism and their mode of operation are far from completely understood, even less is known of their distribution and function in the plant world, and no information is available on the mode of disposal of synephrine and related phenolic amines in nature. Hence, the following studies on the distribution of synephrine and related compounds in Rutaceous plants and on the isolation and characterization of microorganisms capable of degrading synephrine in nature were carried out.

Chromatographic analyses of the phenolic amine fractions derived from methanolic extracts of the leaves of various locally growing Rutaceous plants revealed that synephrine is the only phenolic amine that accumulates in appreciable amounts in Citrus aurantium, C. medica, C. pennivesiculata and Murraya koenigii. No detectable levels of phenolic amines could be obtained from the leaves of Aegle marmelos, C. decumana, C. limetta, C. paradisi, Murraya exotica and Ruta graveolens.

C. medica and M. koenigii were found to contain 276 mg/kg and 306 mg/kg synephrine (fresh weight leaves), respectively. Synephrine isolated from these two sources was characterized by comparing its ultraviolet and infrared spectra with those of an authentic sample. Periodate oxidation of both the isolated and authentic samples of synephrine yielded p-hydroxybenzaldehyde, which was also characterized.

Microorganisms capable of degrading dl-synephrine were isolated from soil samples collected from Citrus gardens by enrichment culture, using synephrine as the sole source of carbon and nitrogen. Based on morphological, biochemical and physiological characteristics, the five isolates obtained were identified as Arthrobacters. Detailed nutritional studies proved that synephrine was the most efficient source of carbon and nitrogen. One isolate, pink in colour and differing from known Arthrobacter species, was assigned the name Arthrobacter synephrinum n. sp.

The washed cell suspension of A. synephrinum readily utilized synephrine. Experiments on synephrine utilization in soil suggested that A. synephrinum may play an important role in the detoxication of synephrine in nature. Synephrine-grown cells rapidly oxidized synephrine, p-hydroxyphenylacetaldehyde, p-hydroxyphenylacetic acid and 3,4-dihydroxyphenylacetic acid without a lag phase. Non-induced cells, however, utilized them only after a considerable lag. Tyramine, catechol, 3,4-dihydroxybenzoic acid and p-hydroxymandelic acid were not oxidized significantly.

Incubation of washed cell suspensions of A. synephrinum with synephrine resulted in the accumulation of p-hydroxyphenylethyl alcohol, p-hydroxyphenylacetaldehyde, p-hydroxyphenylacetic acid and 3,4-dihydroxyphenylacetic acid in the medium. Cell-free extracts converted synephrine to p-hydroxyphenylacetaldehyde and oxidized externally added p-hydroxyphenylacetaldehyde into p-hydroxyphenylacetic acid in the presence of NAD?. An aromatic oxygenase, obtained in a cell-free system, degraded 3,4-dihydroxyphenylacetic acid by meta-cleavage.

On the basis of these studies, a new pathway for the degradation of synephrine was proposed:

Synephrine ? p-Hydroxyphenylacetaldehyde ? p-Hydroxyphenylacetic acid ? 3,4-Dihydroxyphenylacetic acid ? Meta-cleavage products

The first enzyme in the pathway, which catalyzes the direct conversion of synephrine into p-hydroxyphenylacetaldehyde, was partially purified from cell-free extracts of A. synephrinum by ammonium sulfate fractionation and Bio-Gel P200 gel filtration. This enzyme catalyzes a novel reaction, previously unknown, which can proceed under anaerobic conditions and may involve dehydration of synephrine followed by elimination of the side chain.

A new sensitive method was developed for estimating p-hydroxyphenylacetaldehyde, exploiting its ability to undergo aldol condensation under alkaline conditions. The enzyme requires a sulfhydryl compound (e.g., GSH or mercaptoethanol) and a divalent cation (Ca²?, Mg²? or Mn²?) for optimal activity. It is highly labile and completely inactivated after 48 hours at 4?°C or –20?°C. The reaction proceeds optimally at pH 8.0 and 30?°C and is linear up to 40 minutes. The enzyme is specific for synephrine and does not metabolize related phenolic amines such as tyramine, N-methyltyramine, dl-octopamine or hordenine.

The enzyme concentration curve showed deviation from linearity at low protein concentrations. The Km for synephrine is 1 mM. The enzyme is sensitive to metal-chelating agents and sulfhydryl inhibitors. The reaction does not require coenzymes such as NADP?, FAD, tetrahydrofolate, thiamine pyrophosphate or pyridoxal phosphate.