JOURNAL OF BAcTrERIoLGY, Oct. 1976, p. 182-191 Vol. 128, No. 1 Copyright 0 1976 American Society for Microbiology Printed in U.S.A. Metabolism of DL-(-+)-Phenylalanine by Aspergillus niger G. KISHORE, M. SUGUMARAN, AND C. S. VAIDYANATHAN* Enzymology Laboratory, Department ofBiochemistry, Indian Institute ofScience, Bangalore 560 012, India Received for publication 26 February 1976 A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified asAspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. 1-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phen- ylpyruvate decarboxylase activities could be demonstrated in the cell-free sys- tem. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenyl- acetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxyman- delate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via 4-hydroxybenzoylformate, 4-hydroxybenzaldehyde, and 4-hydroxybenzoate. Microbial metabolism ofaromatic compounds rophosphate (TPP), nicotinamide adeninine dinu- has been the subject of intense research for cleotide phosphate (NADP), flavine adenine dinu- several years, as evidenced by the review arti- cleotide (FAD), riboflavine 5'-phosphate (FMN), pyridoxal phosphate (PALPO), phenazine methosul- cles that have appeared in literature (4, 7, 22, fate (PMS), glucose-6-phosphate, and glucose-6- 23). However, studies on the fungal degrada- phosphate dehydrogenase were from Sigma Chemi- tion of phenylalanine have received only scant cal Co., St. Louis, Mo. 4-Hydroxybenzoic acid was attention. The major fate of phenylalanine, in supplied by K & K Laboratories, Inc., California. most of the fungi studied, is its degradation to All the other chemicals used were of analytical cinnamic acid and to phenylpyruvic acid (10, grade available commercially and purified when 14-16, 22, 23). Cinnamate thus formed is con- necessary. verted to protocatechuate through benzoate, Organism. A. niger isolated from the soil and is converted to homo- identified by N. G. K. Karnath, of the Microbiology whereas phenylpyruvate and Pharmacology Laboratory of the Indian Insti- gentisate before ring cleavage (22, 23). Among tute of Science, was used throughout the course of the other pathways suggested for the phenylal- these investigations. anine metabolism is the one involving 4-hy- Cultivation. The organism was grown on a modi- droxymandelate (3, 18). Perrin and Towers (18) fied synthetic medium (1) containing the following have detected radioactive 4-hydroxymandelate nutrients: glucose, 50 g; KNO3, 5 g; KH2PO4, 2.5 g; in the culture filtrates of Polyporous hispidus MgSO4 * 7H20, 1 g; Na2SO4, 1 g; DL-phenylalanine, 1 grown on phenylalanine or tyrosine. On the g (as the inducer); FeCl3 * 6H20, 20 mg; ZnSO4 * 7H20, basis of2,5-dihydroxybenzoylformate formation 10 mg; MnSO4 * 4H20, 3 mg; Na2MoO4 * 2H20, 1.5 mg; from both 4-hydroxymandelate and 4-hydroxy- and CuSO4 * 5H20, 1 mg, in 1 liter of water. The pH of the medium was adjusted to 5.5 with 3 N NaOH. phenylacetate, Crowden (3) has suggested that The medium was distributed in 500-ml conical flasks 4-hydroxymandelate is formed from 4-hydroxy- (60 ml per flask) and sterilized by autoclaving at phenylacetate. In a preliminary communica- 120°C) for 15 mins. Stock cultures were maintained tion (12), we have reported the isolation and on slants of the same medium solidified with 2% identification of 4-hydroxymandelate as a key agar. The flasks were inoculated with a heavy sus- intermediate in the catabolism of phenylala- pension of spores in sterile water, obtained from nine by Aspergillus niger. In this paper we agar slants 96 or more h old and incubated at 30°C wish to report our detailed investigations on for various intervals of time, ranging from 26 to 60 the pathway. h, depending upon the experiment to be done. The mycelia were harvested before sporulation after de- canting out the unspent medium and washing it MATERIALS AND METHODS with distilled water. The mycelia were then Chemicals. DL-Phenylalanine, sodium phenylpy- squeezed and dried between the foldings of a blot- ruvate, 2-hydroxyphenylacetic acid, 4-hydroxyphen- ting paper. The dried mycelia could be used directly ylacetic acid, 4-hydroxymandelic acid (DL), 3,4-dihy- for preparation of enzymes and in vivo experiments droxybenzoic acid, homogentisic acid, thiamine py- or stored at -20°C until required. 182 VOL. 128, 1976 DL-PHENYLALANINE METABOLISM IN A. NIGER 183 In vivo experiments. Mycelial felts of A. niger, after 4 h were isolated in a manner similar to that during various phases of growth, were taken, described under in vivo experiments. The isolated washed as described above, and suspended in solu- phenolics were subjected to chromatography on tions containing 1 mg of the metabolite of interest Whatman no. 3 filter paper, using the solvent sys- per ml in 0.025 M sodium phosphate buffer, pH 7.0 tem A/B, and the chromatogram was kept over an (hereafter referred to as the "replacement medium"). X-ray film for 2 weeks. The film was then developed, The culture suspensions were placed on a rotary and the radioactive zones were localized. shaker at 30°C for 4 to 6 h, and the products formed Estimation of concentrations of phenolic com- were analyzed. pounds during various phases of growth. The con- Isolation of phenolic compounds. For the isola- centrations of phenolic metabolites formed during tion of phenolic compounds from the aqueous solu- various phases of growth were determined as fol- tion, the mycelial felts were removed and the me- lows. The phenolic compounds extracted from the dium was filtered through glass wool. The filtrate culture medium were subjected to quantitative two- was acidified to pH 2 with 2 N HCI and extracted dimensional chromatography using Whatman no. 3 repeatedly with peroxide-free distilled diethylether. paper and solvent system A/B. The individual com- The ethereal layers were pooled and resolved into pounds were eluted from the chromatogram using the acidic and neutral fractions by extraction with hot ether. After evaporating the ether, they were 5% NaHCO3. The acidic fraction was reextracted used for estimation. Determinations of 2- and 4- into ether after acidification to neutralize the bicar- hydroxyphenylacetic acids were done by the 4-ni- bonate. Both the fractions were dried over anhy- troaniline method described under Assay of pheny- drous sodium sulfate, concentrated, and subjected to lacetate hydroxylase. 3,4-Dihydroxybenzoic acid chromatography. was estimated by the method ofNair and Vaidyana- Chromatography. The following solvent systems than (17), and 2,5-dihydroxyphenylacetic acid was were used for the chromatographic separation (as- estimated, by the method of Fellman et al. (5). cending) and identification of the phenolic com- Preparation of cell-free extract of A. niger. The pounds: solvent A; benzene-acetic acid-water (10:7:3 cell-free extract ofA. niger was obtained as follows. [vol/vol], organic phase); solvent B, formic acid-wa- Mycelial felts, 10 g, were mixed with an equal ter (2:98 [vol/vol1; and solvent C, isopropanol-am- weight ofglass powder and ground to a fine slurry in monia-water (20:1:2 [vol/vol]). The chromatograms a mortar. The slurry was uniformly suspended in 30 were run at 30°C on Whatman no. 3 filter paper. ml of 0.025 M sodium phosphate buffer, pH 7.0, and Detection. Aromatic compounds were located on the cell debris as well as insoluble particles were the chromatograms by ultraviolet (UV) light. Phe- removed by centrifugation at 27,000 x g for 30 min. nolic compounds could be identified by spraying The supernatant obtained was designated as the either with (i) freshly prepared, diazotized 4-ni- "crude extract" and used as an enzyme source. For troaniline reagent (3 ml of 0.3% solution of 4-ni- preparation of the particulate fraction, the crude troaniline in 0.8 N HCI plus 0.5 ml of 5% sodium extract was subjected to ultracentrifugation at nitrite, mixed before use) followed by alkali or (ii) a 100,000 x g for 60 min. The supernatant obtained 1% ferric chloride-ferricyanide mixture (1:1). Car- was designated as the "soluble fraction," and the bonyl compounds could be recognized by their reac- pellet, after suspension in 0.025 M sodium phos- tion with 2,4-dinitrophenyl hydrazine (0.1% 2,4-di- phate buffer, was referred to as the "particulate nitrophenylhydrazine in 2 N HCI) followed by alkali. fraction." The various phenolic compounds that were re- Unit of enzyme activity. One unit of enzyme ac- solved by paper chromatography were eluted from tivity is defined as the amount that catalyzes the the filter paper with ether. The ether extract was transformation of 1 umol of the substrate or the concentrated and subjected to repeated rechroma- formation of 1 j.mol ofthe product per min under the tography so as to isolate a pure material. conditions ofthe assay. Specific activity is expressed UV spectra. A Cary-14 recording spectrophotome- as milliunits of enzyme activity per milligram of ter was used for obtaining spectra of the various protein. compounds in both the UV and visible