I N C O R P O R A T I O N OF A C E T A T E I N T O TRICHOPHYTON RUBRUM PHENYLALANINE
by R . A . ZUSSMAN1), E . E . VICItER & I.
LYON 2)
(12.III.196S) The pentose phosphate and shikimic acid pathways do not appear to be involved in phenylalanine biosynthesis in Trich@hyton rubrum (ZussMAN, VICttER & LYON, 1967, and ZUSSMAN, VICHER & LYON 1969). This report presents evidence that acetate is involved and shows the effect of several specifically pertinent organic compounds upon the incorporation of acetate into fungal phenylalanine. The Millipore filter culture technique was employed (ZusSMAN, 1959). An amino acid-free medium (GEoRG & CAMP, 1957) containing sodium acetate-l-C 1~ was inoculated with a CDC strain of T. rubrum and incubated at 80° C for 25 days. Cultures were grown in one liter chambers containing the vapors of 10-aM acetonitrile, acetylene, butadiene, cyclohexane, cyclohexanol, cyclohexanone, or cyclohexene. Control cultures were grown in air and chamber-confined air. Fungal protein was extracted, hydrolyzed, and chromatographed by methods previously described (ZussMAN, VICHI~R& LYON, 1967) and the specific activity of isolated phenylalanine was determined by techniques cited therein. Results appear in the table. Acetate-l-C .4 was incorporated into fungal phenylalanine (see "Control culture b"). This amino acid contained 2.9 % of the specific activity (total carbon) of substrate acetate-l-C 14. It may also be observed that acetonitrile and cyclohexanone markedly increased the incorporation of acetate. The reasons underlying these results are not known. One may postulate poisoning of acetate-utilizing systems by either of these chemically reactive compounds thereby shunting acetate into phenylalanine biosynthesis. Secondly, if the acetonitrile carbon skeleton is directly incorporated into metabolites in positions normally occupied by acetate, a "sparing" of acetate for phenylalanine synthesis may also be considered. A third possibility under the given experimental 1) Present address: Abbott Laboratories Scientific Division, North Chicago, Illinois. 2) Present address: Bermington College, Bennington, Vermont.
A C E T A T E IN T. R U B R U M P H E N Y L A L A N I N E
TABLE
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Influence of Organic Compounds upon lhe Incorporation o / A cetate-l-C 1~ iuto Trichophyton rubrum Phenylalanine Organic compound (vapor) (10-a M) in atmosphere
Pheny]alanine specific activity, relative (total carbon)
Acetylene Acetonitrile 13utadiene Cyclohexa~e Cyclohexanol Cyelohexanone Cyclohexene Control a (grown in air) Control b (grown in 1 liter Chamber)
53 % 455 83 123 Growth completely inhibited 1127 135 100 145
conditions may be that, if the acetonitrile carbon skeleton itself enters the phenytalanine ring, it may increase the entry of acetate into the ring. In this instance, both the rate of phenylalanine biosynthesis and its specific activity would increase. It would be interesting to determine if acetonitrile can provide carbon for the phenylalanine ring or for other metabolites; such data would be useful for the interpretation of these results. It is of interest to note that cyclohexanol inhibited the growth of Trich@hyton rubrum completely. ]~HRENSVAARD & ]~EIO (1953), GATENBECK (1958), (1960) have reported that acetate is involved in the synthesis of a variety of fungal products, excluding the aromatic amino acids which are usually derived via the shikimic acid pathway. One might hypothesize that, in T. rubrum, two-carbon fragments derived from acetate fuse to form aromatic rings leading to the synthesis of phenylalanine and, possibly, tyrosine. The mechanism may be similar to that required for the synthesis of the "Raistrick compounds" in saprophytic fungi. This avenue of inquiry will be pursued further with
Trich@hyton mbrum. Acknowledgement This study was supported in part by Grant No. E-3750, C1, National Institutes of Health, United States Public Health Service.
References EHRENSVAARD, G. & L. REIO. 1953. The Formation of Tyrosine in Escherichia coli and Neurospora crassa Grown on Isotope-labeled Acetate as the Sole Carbon Source. Arkiv Kemi. 5: 229--234. GATX~BECK, S. 1958. Incorporation of Labeled Acetate in Emodin in ]Penicillin islandicum. Acta chem. Scand. 12: 1211--1214.
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GATENBI~CK, S. 1960. Studies on the Biosynthesis of Anthraquinones in Lower Fungi. Svensk Kern. Tidskr. 72: 188--203. GV2ORG, L. K. & L. CA~P. 1957. Routine Nutritional Tests for the Identification of Dermatophytes. J. Bact. 74: 113--121. Zuss~IAN, R. A. 1959. Pigment-forming h