1898
COMMUNICATIONS TO THE EDITOR
Vol. 73
that the active substance was L-histidinol' (L-2amino-3- [4(or 5)-imidaz~lyl]-l-propanol). The identity was established by comparison of the isolated dihydrochloride and dipicrate with a synthetic sample of L-histidinol dihydrochloride2and the dipicrate prepared from it. The corresponding melting points were identical and mixed melting points undepressed. Natural and synthetic L-histidinol dihydrochloride give the same response (equivalent to that of 75% of their weight of Lhistidine dihydrochlorjde) with strain 26-24D 1, which was used as assay organism in the isolation. Excretion of L-histidinol by one mutant and utilization by others suggest that this compound is an intermediate in the biosynthesis of histidine in E . coli. That L-histidinol is utilized slowly by 26-24 does not invalidate this interpretation, since an analogous phenomenon encountered with shikimic acid, a common precursor of aromatic metabolites, has been e ~ p l a i n e d . ~Factors affecting the rate of utilization of L-histidinol are being further investigated.
Solutions of thiaminase were made from fresh carp viscera as previously These were incubated for one hour at 30' with the 2 substrates, viz., the thiazolium salt and the amine. PAB yielded pteroic acid, and PABG gave pteroylglutamic acid, as judged microbiologically. Either substrate alone with the enzyme gave no new folic acid. U'ithout enzyme, the 2 substrates yielded small amounts of folic acid, but this was greatly augmented by the enzyme. Thus 4 cc. of carp extract plus 3 mg. each of PAB and thiazolium salt yielded 10 gamma pteroic acid; enzyme blank 1.8 gamma; substrate blank 0.6 gamma. The pH dependence and the need for a dialyzable component were similar to those for thiaminase activity. The specificity of the enzyme was directed to the thiazolium part of Qe molecule, because no synthesis of folic acid was observed with the corresponding pyridinium salt,6 which can be used in the chemical synthesis of this ita am in.^ Because the natural occurrence of the thiazolium U. S. PUBLICHEALTHSERVICE HENRYJ. VOGEL' salt is unknown there is no proof that this is the TUBERCULOSIS RESJMRCH LABORATORY BERNARD D. DAVIS mode of biosynthesis of folic acid. Rather, it is CORNELLUNIVERSITY MEDICAL COLLEGE offered as experimental evidence for a new kind of SEWYORK 21, S. Y. ELIZABETH S. NISGIOLI biosynthetical mechanism in which the driving RECEIVED MARCH16, 1951 force resides not in a phosphate bond, but in a (1) P. Karrer, M. Suter and P Waser, H c h . Chrm Arra, 38, 1936 quaternary ammonium ion which is reduced to a m p. 193-195' (uncor 1, (1949). Reported for dihydrochloride tertiary amine during the reaction. From the [ c z ~ * ~-3D 9S0 (water). existing information about relationships of folic (2) Generously furnished by Professor P. Karrer. acid and vitamin B12, it is quite possible that the (3) B. D. Davis, J . Biol. Chsm., in press (4) U S Public Health Service Research Fellow. dialyzable coenzyme of carp thiaminase may contain this vitamin, and efforts t o learn about this are in progress. ENZYMATIC SYNTHESIS OF FOLIC ACID BY THE ACTION OF CARP THIAMINASE
Sir :
(3) D. W. Woolley, J . Bzol. Chcm , 141, 997 (1941). (6) Kindly supplied by the American Cyanamid Company
Carp thiaminase destroys thiamine by cleaving FROM D. W. WOOLLEY THEROCKEFELLER INSTITUTE off the thiazole moiety and uniting the pyrimidine FOR MEDICAL RESEARCH portion of the vitamin to some unknown substance hTE\i' YORK,AT. Y. RECEIVED MARCH 9, 1961 in the enzyme preparation.' Sealock and Davis2 have shown that this latter material can be replaced by nitro-aniline, which is alkylated on the amino group by the pprimidylmethyl part of thiamine. BIOLOGICAL PRECURSORS OF THE PYRIMIDINES They have suggested the similarity of this reaction Sir : to transmethylation. If this view be correct, then Lactobacillus bulgaricus 09 has been found to reother amines might be alkylated by other suitably quire either orotic acid or ureidosuccinic acid as an constituted quaternary salts when catalyzed by essential growth factor.1,2Jn4 The following exthis enzyme. I n this way certain other meta- periments with Lactobacillus bulgaricus 09 are conbolically essential substances might be formed, cerned with the role of these compounds in the biosuch as pteroic acid and its derivatives, which are genesis of the pyrimidine components of ribonuamines alkylated with a substituted methyl group. cleic acid. 2-Amino-4-hydroxy-6-pteridylmethyl(4'-methylLactobacillus bulgaricus 09 was grown in 500 ml. 3'-hydroxyethylthiazolium) bromide (a pteridine amounts of pyrimidine-free basal medium ( 2 ) conanalog of thiamine) was formed by the stepwise re- taining, in the first experiment, 5 mg. of added action of cu,&dibromopropionaldehyde with "thi- orotic acid (5) labelled in position 2 with CI4 and, amine thiazole"3 and then with 2,4,5-triamino-6- in the second experiment, with 15 mg. of added hydroxypyrimidine as in a related synthesis leading m-ureidosuccinic acid5 (aseptic addition) labelled to folic acid4; although the compound was rather (1) L. D. Wright, J. W. Huff, H. R. Skeggs, K. A. Valentik and D unstable, it was obtained analytically pure. K.B o s s i r a r d t , T ~JOURNAL, ~s 72,2312(1950). (1) L 0 Krampitz and D W Woollej. J Bgol Chein , 168, 9 (1944). (2) R R Sealock and N C Dams, r b i d , 177, 987 11949) ' 3 ) "Thiamne thiazole" was kindly supplied by Dr G A Emerson f l i M E Hultquist, E Kuh, D B. Cosulich, XI 1 Fahrenbach, L H Korthey, D. R Seeger, J P Sickels T X i Smith, J r R R .Inbier, J H Boothe, B L Hutchlngs, J H Zlowat, J k m b I L R stokstad Y SuhbaRow and C W \X'iIl-r. 'Ii i i i h Y 4 nd % I Yzrfiglrrnenl 48, 1 (1917)
(2) L. D.Wright, K. A. Valentik, D. S. Spicer, J. W. Huff and H. R . Skeggs, Proc. Soc. Expll. B i d . & M c d . , TS, 293 (1950). (3) 0. P. Wieland, J. Avener, E. M. Boggiano, N. Bohonos, B . L. Hutchings and J. H. Williams, J. Bid. Ckcm., 186,737 (1950). (4) The microbiological activity previously reported for 5-(carboxyniethy1idine)-hydantoin could not be confirmed with more carefully prepared preparations. Evidence is now available that this activity was due to contaminating orotic acid. 1%); J. 1'. T p c arid T I I;.Mitc-hell,THIS J O U R K A L , 69, 1382( 19t7).
