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The isolation of D -valine from Penicillium chrysogenum
PRELIMINARY NOTES
377
The isolation of D-valine from Penicillium chrysogenurn During the course of work on the identification of possible intermediates in penicillin biosynthesis, we have isolated, from the mycelium of Penicillium chrysogem~m, several compounds containing cystine/cysteine and valine. Since these amino acids are precursors of penicillin 1, intermediates in the biosynthetic pathway must contain then in some form. The valine moiety in the final penicillin molecule has the o-configuration although 5-valine is the probable ultimate precursor 2, a. It was of interest, therefore, to examine the configuration of the valine derived from the above compounds. Mycelium of P. chrysogenum strain Wis. 5I-2oF3 was grown in the medium of JAaVlS .aND JOHNSOI~4, and harvested and washed as previously described 5. The washed mycelium was resuspended in o.oi M phosphate buffer, p H 7.0, containing 0.o25 g/1 potassium phenylacetate (ioo g wet mycelial pad in 8oo ml of liquid in terL 5oo-ml flasks), and shaken for I h at 24 °. These conditions permit the continued synthesis of benzylpenicillin 5. The mycelium was then washed again with three changes of water at 2 ° . The aim of this washing at reduced temperature was to minimize further metabolism of any accumulated intracellular intermediates. After the final washing, the mycelium was subjected to mild acid hydrolysis (8o0 ml I N HC1, 56°, 2 h), filtered, and the cooled filtrate extracted with diethyl ett:er. Some purification of the extract was effected by extraction into aqueous but:fer at p H 7 and back into ether at pH 2. The extract was then evaporated to about 0. 5 ml, and portions were fractionated by paper chromatography (Whatman No. i paper, butanol-acetic acid-water (lOO:24:1oo , v/v/v), samples neutralized with ammonia vapor before running), and the paper sprayed with anamoniacal AgNOa. The most prominent spots which appeared were colorless against the brown background and had RE values of o.61, 0.79 and 0.86; they gave no color reaction wilh ninhydrin. Complete hydrolysis of the substances eluted from these spots yielded the same mixture of amino acids in each case, as judged by conventional paper chromatography and spraying with ninhydrin. The amino acids appeared to be valine, cystine/cysteine, glycine and leucine. Although attempts were made to demonstrate phenylacetic acid the side-chain precursor of penicillin--in the hydrolysates, this has not yet been accomplished to our satisfaction. It is presumed that the three substances yielding amino acids on further hydrolysis are peptides. The yield of valine from IOO g of mycelial pad (IO g dry wt.) was approximately 50/xg. Because of the small quantities of material available, the following micro methods were used to examine the configuration of the isolated valine: I. Paper chromatography. Conventional chromatography, with the conditions detailed above, showed that I)- and L-valine differed slightly but consistently in their RE values. 2. Microbiological assay. Species of lactobacilli differ in their growth response to the optical isomers of amino acids% ]'he responses (acid production after 3-days' incubation) of Lactobacillus arabinosus 17- 5 and L. fermenti ATCC 9338 to the isolated valine and to graded amounts of D- and L-valine were measured in a synthetic valine assay medium. 3. D-Amino acid oxidase. The isolated valine was treated with crude D-amino 23iochim. Biophys. Acta, 42 (196o) 377-378
378
PRELIMINARY
NOTES
acid oxidase (Sigma Chemical Co.) of k n o w n a ct i v i t y , in a 0.02 M p y r o p h o s p h a t e buffer, p H 8. 3. Th e a m m o n i a liberated during 5-min incubation was d e t e r m i n e d colorimetrically with Nessler's reagent. K n o w n q u a n t i t i e s of D- and L-valine were similarly treated. 4. Niuhydrin reaction. The t o t a l valine was d e t e r m i n e d by the q u a n t i t a t i v e nin h y d r i n reactionL T h e results of these four d e t e r m i n a t i o n s are shown in Table I. TABI~E l R E S P O N S E S OF L-, 17)- AND ISOI,ATI~2I) V A L I N E TO VARIOUS T E ST S 3fctkod:
I RF ( B u t a n o l acetic acid)
L-valine D-valine Isolated valine
o.49 o.51 o. 51
2 L. arabl)~osus
L. fl'rmenti
+ - -
+ + +
]
4
o-amino acid oxidase
Ninhydriu
-!+
Q u a n t i t a t i v e l y , the positive responses of the isolated valine were similar; for example, a single sample assayed 13 ( ~ 3) /xg, ~2 ( ~ 3) /*g and 12 ( ~ I) #g by m e t h o d s 2, 3 and 4 respectively, indicating the absence of L-valine within e x p e r i m e n t a l error. Thus all the valine in each of the three peptides was D-valine. T h e presence of peptides of D-valine in a penicillin-producing organism m i g h t be expected, in v i e w of the s t r u c tu r e and origin of the penicillin molecule. H o w e v e r , this is the first r e p o r t of the isolation of D-valine from the m y c e l i u m of P. chrysogenmn, where it has been claimed to be absent s. T h e existence of intracellular, ether-soluble peptides c o n t a i n i n g cystine/cysteine and D-valine has i m p o r t a n t implications for theories of the m e c h a n i s m of penicillin biosynthesis. These will be discussed in a more detailed account to be published elsewhere. This work has been s u p p o r t e d by the Australian N a t i o n a l H e a l t h and Medical Research Council. The authors wish to t h a n k Drs. S. A. KOSER, M. J. JOHNSON" and H. C. LICHSTEIN for supplying cultures.
Department of Bacteriology, University oJ Oueensland, Brisbane (Australia)
\V. J. HALLII)AY [~}ETTY E. BRA1)FIELD M. K. SHAW
1 H. R. V. ARNSTEINAND P. T. (;RANT, Biochem. J., 57 (I954) 353, 300. '2 C. M. STEVENS,E. INAMINEAND C. \V. DELONG,J. Biol. Chem., 219 (1956) 4o5 . a H. It. V. ARNSTEINAND H. -~IARGREITER, Biochem. J., 68 (1958) 339. F. G. JARVISAND M. J. JOHNSON,J. Bacleriol., 59 U95 o) 5 J. 5 F. 14.. (;RAU AND \V. J . HALLIDAY,Biochem, .]., 09 (I958) 205. 6 S. A. KOSER AND J . 1,. THOMAS,./. Bacteriol., 73 (1957) 477. v S. MOORE ANI) \V. H. STEIN',J. Biol. Chem., i76 (1948) 367 . s C. M. STEVENS, P. E. HALPERN AND R. P. GIGGER,ft. Biol. Chem., 19o (195I) 7o5. R e c e i v e d .June 7th, I96O
Biochim. Biophys..t eta, 42 (I 96o) 377-378
377
The isolation of D-valine from Penicillium chrysogenurn During the course of work on the identification of possible intermediates in penicillin biosynthesis, we have isolated, from the mycelium of Penicillium chrysogem~m, several compounds containing cystine/cysteine and valine. Since these amino acids are precursors of penicillin 1, intermediates in the biosynthetic pathway must contain then in some form. The valine moiety in the final penicillin molecule has the o-configuration although 5-valine is the probable ultimate precursor 2, a. It was of interest, therefore, to examine the configuration of the valine derived from the above compounds. Mycelium of P. chrysogenum strain Wis. 5I-2oF3 was grown in the medium of JAaVlS .aND JOHNSOI~4, and harvested and washed as previously described 5. The washed mycelium was resuspended in o.oi M phosphate buffer, p H 7.0, containing 0.o25 g/1 potassium phenylacetate (ioo g wet mycelial pad in 8oo ml of liquid in terL 5oo-ml flasks), and shaken for I h at 24 °. These conditions permit the continued synthesis of benzylpenicillin 5. The mycelium was then washed again with three changes of water at 2 ° . The aim of this washing at reduced temperature was to minimize further metabolism of any accumulated intracellular intermediates. After the final washing, the mycelium was subjected to mild acid hydrolysis (8o0 ml I N HC1, 56°, 2 h), filtered, and the cooled filtrate extracted with diethyl ett:er. Some purification of the extract was effected by extraction into aqueous but:fer at p H 7 and back into ether at pH 2. The extract was then evaporated to about 0. 5 ml, and portions were fractionated by paper chromatography (Whatman No. i paper, butanol-acetic acid-water (lOO:24:1oo , v/v/v), samples neutralized with ammonia vapor before running), and the paper sprayed with anamoniacal AgNOa. The most prominent spots which appeared were colorless against the brown background and had RE values of o.61, 0.79 and 0.86; they gave no color reaction wilh ninhydrin. Complete hydrolysis of the substances eluted from these spots yielded the same mixture of amino acids in each case, as judged by conventional paper chromatography and spraying with ninhydrin. The amino acids appeared to be valine, cystine/cysteine, glycine and leucine. Although attempts were made to demonstrate phenylacetic acid the side-chain precursor of penicillin--in the hydrolysates, this has not yet been accomplished to our satisfaction. It is presumed that the three substances yielding amino acids on further hydrolysis are peptides. The yield of valine from IOO g of mycelial pad (IO g dry wt.) was approximately 50/xg. Because of the small quantities of material available, the following micro methods were used to examine the configuration of the isolated valine: I. Paper chromatography. Conventional chromatography, with the conditions detailed above, showed that I)- and L-valine differed slightly but consistently in their RE values. 2. Microbiological assay. Species of lactobacilli differ in their growth response to the optical isomers of amino acids% ]'he responses (acid production after 3-days' incubation) of Lactobacillus arabinosus 17- 5 and L. fermenti ATCC 9338 to the isolated valine and to graded amounts of D- and L-valine were measured in a synthetic valine assay medium. 3. D-Amino acid oxidase. The isolated valine was treated with crude D-amino 23iochim. Biophys. Acta, 42 (196o) 377-378
378
PRELIMINARY
NOTES
acid oxidase (Sigma Chemical Co.) of k n o w n a ct i v i t y , in a 0.02 M p y r o p h o s p h a t e buffer, p H 8. 3. Th e a m m o n i a liberated during 5-min incubation was d e t e r m i n e d colorimetrically with Nessler's reagent. K n o w n q u a n t i t i e s of D- and L-valine were similarly treated. 4. Niuhydrin reaction. The t o t a l valine was d e t e r m i n e d by the q u a n t i t a t i v e nin h y d r i n reactionL T h e results of these four d e t e r m i n a t i o n s are shown in Table I. TABI~E l R E S P O N S E S OF L-, 17)- AND ISOI,ATI~2I) V A L I N E TO VARIOUS T E ST S 3fctkod:
I RF ( B u t a n o l acetic acid)
L-valine D-valine Isolated valine
o.49 o.51 o. 51
2 L. arabl)~osus
L. fl'rmenti
+ - -
+ + +
]
4
o-amino acid oxidase
Ninhydriu
-!+
Q u a n t i t a t i v e l y , the positive responses of the isolated valine were similar; for example, a single sample assayed 13 ( ~ 3) /xg, ~2 ( ~ 3) /*g and 12 ( ~ I) #g by m e t h o d s 2, 3 and 4 respectively, indicating the absence of L-valine within e x p e r i m e n t a l error. Thus all the valine in each of the three peptides was D-valine. T h e presence of peptides of D-valine in a penicillin-producing organism m i g h t be expected, in v i e w of the s t r u c tu r e and origin of the penicillin molecule. H o w e v e r , this is the first r e p o r t of the isolation of D-valine from the m y c e l i u m of P. chrysogenmn, where it has been claimed to be absent s. T h e existence of intracellular, ether-soluble peptides c o n t a i n i n g cystine/cysteine and D-valine has i m p o r t a n t implications for theories of the m e c h a n i s m of penicillin biosynthesis. These will be discussed in a more detailed account to be published elsewhere. This work has been s u p p o r t e d by the Australian N a t i o n a l H e a l t h and Medical Research Council. The authors wish to t h a n k Drs. S. A. KOSER, M. J. JOHNSON" and H. C. LICHSTEIN for supplying cultures.
Department of Bacteriology, University oJ Oueensland, Brisbane (Australia)
\V. J. HALLII)AY [~}ETTY E. BRA1)FIELD M. K. SHAW
1 H. R. V. ARNSTEINAND P. T. (;RANT, Biochem. J., 57 (I954) 353, 300. '2 C. M. STEVENS,E. INAMINEAND C. \V. DELONG,J. Biol. Chem., 219 (1956) 4o5 . a H. It. V. ARNSTEINAND H. -~IARGREITER, Biochem. J., 68 (1958) 339. F. G. JARVISAND M. J. JOHNSON,J. Bacleriol., 59 U95 o) 5 J. 5 F. 14.. (;RAU AND \V. J . HALLIDAY,Biochem, .]., 09 (I958) 205. 6 S. A. KOSER AND J . 1,. THOMAS,./. Bacteriol., 73 (1957) 477. v S. MOORE ANI) \V. H. STEIN',J. Biol. Chem., i76 (1948) 367 . s C. M. STEVENS, P. E. HALPERN AND R. P. GIGGER,ft. Biol. Chem., 19o (195I) 7o5. R e c e i v e d .June 7th, I96O
Biochim. Biophys..t eta, 42 (I 96o) 377-378