Metabolism of N-carbobenzoxyl-L-tryptophan by Chromobacterium violaceum

133

Biochimica et Biophysica Acta, 385 (1975) 133--144

© Elsevier Scientific Publishing Company, Amsterdam-- Printed in The Netherlands

BBA 27600 METABOLISM OF N-CARBOBENZOXYL-L-TRYPTOPHAN BY CHR OMOBA C T E R I U M VIO LA CE UM

P A T R I C K J. DAVIS, M A R K

GUSTAFSON

and J O H N P. R O S A Z Z A

Division of Medicinal Chemistry and Natural Products, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242 (U.S.A.) (Received September 9th, 1974)

Summary Chromobacterium violaceum (ATCC 12472) metabolizes N-carbobenzoxyl-L-tryptophan into its 2',3'~dehydro
Introduction The bacterium Chromobacterium violaceum shows great versatility in metabolizing L-tryptophan. Among the products formed by this organism are the pigment violacein [1--3]; transamination products, indole-3-pyruvic acid and indole-3-acetic acid [4] ; and 5-hydroxy- [5,6], and 6-hydroxytryptophan [7]. 5-Hydroxytryptophan possesses potential for use as a drug [8,9]. The chemical synthesis of this compound is lengthy and costly. An enzymatic synthesis of 5-hydroxytryptophan and other tryptophan derivatives has been reported [10]. We attempted to utilize the hydroxylating capacity of C. violaceum in a microbiological synthesis of 5-hydroxytryptophan using N-carbobenzoxyl-L-tryptophan (Cbz-Trp, I) as a substrate. The Cbz-Trp substrate was used, because it was assumed that by blocking the amino group of tryptophan, it would be possible to block metabolic pathways other than tryptophan hydroxylase in the organism. This approach has previously been taken in the microbiological preparation of L-3,4
134

~

CH2

H

(I)

/COOH

~

"~

CBZ CBZ=

-- ~--O--CH~

C H..~

~H

COOH

CBZ

(11)

Fig. 1. Conversion of N-caxbobenzoxyl-L-tryptophan (I) to N-carbobenzoxyl-2',3'-tryptophan (II).

When Cbz-Trp was incubated with a growing culture of C. violaceum, no 5-hydroxylase activity was observed. However, an unidentified metabolite was found to accumulate in large amounts in fermentation media. This paper describes aspects of the production, isolation and characterization of the metabolite as 2',3°-dehydro-N-carbobenzoxytryptophan (II) (Fig. 1 ). Experimental and Results

General Melting points were determined in open-ended capillaries and are corrected. Infrared spectra were obtained on a Beckman-10 spectrophotometer in KBR discs. Ultraviolet spectra were taken on a Pye-Unicam SP1800 instrument in methanol. Nuclear magnetic resonance spectra were obtained with a Varian T-60 instrument in [2 H6 ] dimethylsulfoxide with tetramethylsilane as an internal standard. Mass spectra were provided through the analytical services of Morgan-Schaeffer Co., Quebec Canada (low resolution), and the Battelle Memorial Institute, Columbus, Ohio (high resolution spectrum). A number of chemicals were purchased or synthesized for this work. The identity of these compounds was confirmed either by melting point determinations, or by the preparation of suitable derivatives. Each c o m p o u n d was also examined by thin-layer chromatography to verify purity. Chemicals utilized were: N-carbobenzoxyl-L-tryptophan (Cbz-Trp), N-acetyl-L-tryptophan, indole-3-propionic acid, indole-3-acrylic acid, N-carbobenzoxyl-L-phenylalanine, N-carbobenzoxyl-L-tyrosine (Sigma); and b-tryptophan (Trp) (Eastman). The following compounds were prepared according to previously published procedures: N-formyl-L-tryptophan [13] ; N-trifluoroacetyl-L-tryptophan [13] ; N-carbobenzoxyl-D-tryptophan [14]. Enzymes used in this work were: L-amino acid oxidase (Sigma, specific activity 5.7 units/mg protein using phenylalanine as substrate), and catalase (Sigma, specific activity 22 000 units/mg protein). Chromatography Chromatographic procedures were used for the analysis of fermentations and for the isolation of fermentation products. Thin-layer chromatography was performed on 0.25 m m thick silica gel GF2 s 4 prepared on glass plates with a Quickfit (Quickfit Industries, London) spreading apparatus. Solvent systems utilized, and mobilities of compounds studied are summarized in Table 1. Column chromatography used in the separation of various microbial metabolites was performed on Baker silica gel (60--200 mesh). Silica gel was

135 normally activated b y heating at 140°C for 4 h prior to use.

General fermentation procedure C. violaceum (ATCC 12472) was used throughout this study. Lyophilized cultures were revived and stored in nutrient broth or in nutrient broth supplemented with 0.1 mg/ml L-tryptophan. Cultures were maintained by transferring them to fresh media at 4--6 week intervals. Cold stored cultures did not remain as viable as those maintained at r o o m temperature. The basal medium used in all fermentations was described b y Mitoma et al. [5]. Fermentations were conducted on rotary shakers (Model G-25, New Brunswick Scientific Co.) operating at 200 rev./min, and 27°C, usually in cotton-plugged, 500-ml Erlenmeyer flasks containing 100 ml of medium. A 2% inoculum from stock cultures was used to initiate Stage I fermentations which were incubated for 72 h before serving as the source of inoculum for Stage II cultures. A 10% inoculum was used to start Stage II cultures which were incubated for 24 h before substrates were added to them. Substrates were dissolved in dimethylformamide, and concentrations were adjusted such that not more than 0.4 ml of dimethylformamide stock solutions were added to each 100 ml of culture medium. Larger scale fermentations were conducted in 10 1 of medium in a Microferm fermentor (MF 114, New Brunswick Scientific Co.) as the Stage II culture. The fermentor was stirred at 350 rev./min, and 27°C, and air was sparged in at 0.4 vol./1 of medium per min. Foaming was controlled by the addition of octanol. For the routine analysis of fermentations, samples of 4 ml were adjusted to pH 2 with 5 M HC1 and extracted with 1 ml of ethyl acetate. Approximately 50 pl of the extracts were spotted on thin-layer chromatography plates, and were co-chromatographed with standards. In most cases, two or three different solvent systems were used to verify the identity of metabolites. Production, isolation and characterization o f the metabolite Initial work indicated that C. violaceum was capable of converting CbzTrp into the unidentified metabolite in approx. 20% yield within 48 h. The metabolite was prepared in amounts sufficient for structure elucidation in the following experiment. The C. violaceum Stage II culture consisted of 25, 500-ml Erlenmeyer flasks. Cbz-Trp in dimethylformamide (2.4 g/9.6 ml) was distributed evenly among them to give a substrate concentration of 1 mg Cbz-Trp/ml culture medium. One flask was kept as a control. According to the thin-layer chromatographic analysis, 40--50% of the substrate had been converted to the u n k n o w n metabolite within 72 h. The 72-h cultures were combined, acidified to pH 2 with 5 M HC1 and extracted three times with 800 ml ether. The ether extracts were combined, dried over anhydrous Na2 SO4 and evaporated to 1.8 g of a dark tarry residue. The tar was taken into ether, and almost immediately yielded a quantity of yellow crystalline material. Successive recrystallization of the crude crystals from methanol gave a total of 290 mg of the metabolite which also crystallized well from benzene.

Spot colors (P - d ~ e t h Y l ~ i n o h e n z

Blue Blue ( g r e e n c e n t e r ) Orange-bro wn Blue-gray Blue Blue Blue Brown

Compound

Tryptophan Indole-3-propionate Indole-3-acrylate Indole-3-pyruvate Indole-3-acetate Indole-3-laetate Cbz-L-tryptophan 2',3t-dehydro-N-Cbz-tryptophan

~i d e h y de-NM~,l 0 2 r e a g e n t )

38

A

23

B

80

C

55 90 25

D

58 33 41 39 33 37 19

E

R f X 1 0 0 in S o l v e n t S y s t e m

77 72

F

21 11

G

28 41

H

T h e s p o t colors o n t h e d e v e l o p e d c h r o m a t o g r a m s w e r e visualized b y s p r a y i n g w i t h 0.1% p - d i m e t h y l a m i n o b e n z a l d e h y d e in 1 . 0 M HC1, d r y i n g p l a t e s w i t h a h e a t gun, a n d s p r a y i n g w i t h 0.1% N a N O 2 s o l u t i o n [ 2 8 ] . S o l v e n t s y s t e m s u s e d ( b y vol. ratios): A, e t h y l a c e t a t e / e t h a n o l / 2 5 % N H 4 O H (9 : 5 : 4); B, c h l o r o f o r m / m e t h a n o l / acetic acid (15 : 5 : 1); C, e t h a n o l / w a t e r (7 : 3); D, c h l o r o f o r m / e t h a n o l / a c e t i c acid ( 9 5 : 5 : 5); E, e t h e r / l i g h t p e t r o l e u m / f o r m i c acid (75 : 50 : 1); F, e t h y l a c e t a t e / i s o p r o p a n o l / 2 5 % N H 4 O H (7 : 5 : 4); G, b e n z e n e / e t h a n o l ( 1 0 : 1); H, b e n z e n e / e t h a n o l (4 : 1).

T H I N - L A Y E R C H R O M A T O G R A P H Y D A T A OF M I S C E L L A N E O U S I N D O L E COMPOUNDS

TABLEI

137 Controls consisting of Cbz-Trp in fermentation medium adjusted to pH 4.0, 6.0 and 7.0 were incubated as described earlier. Thin-layer chromatographic analysis resulted only in the recovery of Cbz-Trp, thus eliminating the possibility that the metabolite represented a fermentation artifact. A larger scale fermentation was conducted in a Microferm fermentor in 10 1 of medium. A total of 5 g of Cbz-Trp in 20 ml of dimethylformamide was added to the culture at 24 h, and the fermentor was harvested after 48 h. After an extraction procedure similar to that described above, 10 g tarry residue was obtained. The tar was dissolved in a minimum volume of ether/light petroleum/formic acid (75 : 50 : 1, b y vol.), and was added to a silica gel column (500 g, 5 × 100 cm) from which 20-ml fractions were collected at a flow rate of 6 ml/min. Fractions 171--385 contained largely the metabolite. These fractions were combined to yield 500 mg of the metabolite.

Physical and spectral properties of the metabolite The analytical sample of the metabolite provided the following physical data: m.p. 218--220°C (decompn); infrared (KBr disc) broad band 2340--3500 cm-' ; 1712, 1656, 1610, 1480, 1410 and 742; NMR ([2 Ha ] dimethylsulfoxide) 85.15 (s, 2,-OCH2-C~Hs ), 7.37 (s, 5, -OCH2 C6Hs ), 7.3 (m, 4, aromatic), 7.8 (m, 3), 8.67 (s, 1, CO-NH-), 11.75 (s, 1, COOH), signals at 11.75 and 8.67 disappeared upon equilibrating samples with 2H2 O; ultraviolet (methanol) kmax230 (E 2.74 " 104), 280 nm (E 8.35 • 103), 333 nm (E 1.80 • 104); addition of HC1 to the sample caused a bathochromic shift of 5 nm of the 333-nm peak, and addition of NaOH caused a hypsochromic shift of 12 nm of the 333-nm peak; mass spectrum, m/e 336.1114, consistent with C~9H16N2 0 4 . The molecular ion was confirmed by preparation of the methyl ester, and subsequent high resolution mass spectrum of this derivative gave m/e 350.1267, consistent with empirical formula C: 0 HI 8 N2 0 4 . Prominent fragment ions in the high resolution mass spectrum of the metabolite are shown in Table 2.

NMR spectral comparison of the metabolite with indole-3-acrylic acid Portions of the NMR spectra of the metabolite and indole-3-acrylic acid which were obtained in [2 H6 ] dimethylsulfoxide are shown in Fig. 2. Signals for the olefinic protons of indole-3-acrylic acid occur as doublets at 6.52 and 7.9 8 (J = 8 cps) for the a-, and ~-protons, respectively. In the NMR spectrum of the metabolite, no a-proton signal exists, and the multiplet at 7.8 8 is simplified, and still integrates for three protons.

Ultraviolet spectral comparisons of the metabolite, Cbz-Trp, and indole3-acrylic acids All c o m p o u n d s were dissolved in methanol in the following concentrations: metabolite, 4.35 • 10 -s M; Cbz-Trp, 9.67 • 10 -s M; and indole-3-acrylic acid, 9.4 • 10 -s M. The results are shown in Fig. 3. Cbz-Trp shows typical indole absorption bands at 276, 283 and 292 nm. Although the metabolite gave a small peak at 280 nm, the most intense absorption was at 333 nm (E = 1.73 • 104 ). Indole-3-acrylic acid exhibited a similar ultraviolet spectrum with a small peak at 276 nm (E = 1.80 • 104 ), and the

138 Table 2. Prominent fragment ions in the high resolution mass spectrum of the metabolite, (H}. l~elative Intensity %

Composition

336. 1114

6.46

C19H16N204

M +"

318. 1061

3.90

CI9H14N20 3

M +" -H20

Z9Z. 1278

52. 16

C]8HI6N2© 2

M +

228. 0551

53. 08

CIzH8N203

M +' - H O C H 2-C6H 5

184.0635

4.45

C ] IH8N20

m/e

Structure or Origin

[.r ~ - -I~- - ' f.J ~ CH\r"~,//CO v

157.0767

47.54

C 1 0H9N

2

-C© 2

"W+ H

NH

{'~'~~CH"~I/H -

-

NH

130. 0650

20. 73

C9H8N

~

I~~ ~ cH2

V - N ¢ H i08.0581

4z. 9o

C7H8 0

91. 0546

i00.00

C7H 7

absorption from 280 consistent the i n d o l e

HOCH2---C6H s

G

m a x i m u m o c c u r r e d at 3 2 6 n m (E = 1 . 9 6 • 104 }. T h e shift in Xm ax t o 3 3 0 n m in b o t h indole-3-acrylic acid and t h e m e t a b o l i t e (II) is w i t h t h e p r e s e n c e o f a d o u b l e b o n d linking t h e c a r b o x y l group w i t h ring [ 1 5 ] .

139

~

~~~C j OOH ('")

I.,,,,I 9

....

I,,,,I 7

$

, 6

I

I

9

,

,

,

,

I

,',

8

,

,

I

7

,

,

,

-

,

I

6

,

6

P,m

Fig. 2. NMR spectral comparisons between the dehydro-metabolite (II), and indole-3-acrylic acid (III).

Decarbobenzoxylation of the metabolite Hydrogenolytic cleavage of the carbobenzoxyl group from the metabolite was accomplished by shaking a suspension of 60 mg of the metabolite and 5 mg of 5% palladium on charcoal in 50 ml of 95% ethanol under 40 lb/inch 2 H2 for 1 h. The metabolite was converted to one major and four minor products, all of which reacted blue with p-dimethylaminobenzaldehyde-NaNO2 reagent (Solvent system A, Table 1). The major reaction product was isolated by preparative thin-layer chromatography using Solvent system A, and was identified as tryptophan by ultraviolet analysis (absorption bands at 276,279 and 289 nm), and by co-chromatography with authentic tryptophan using Solvent systems A, B and C (Table 1). Indole-3-pyruvic acid, indole-3-1actic acid, and indole3-acetic acid were also identified in the hydrogenation reaction mixture by co-chromatography with authentic standards (Solvent systems D and E). Metabolism of indole-3-propionic acid by C. violaceum Indole-3-propionate was added to 24-h Stage II cultures to a final concentration of 0.5 mg/ml of culture medium. Samples were extracted and examined by thin-layer chromatography in the usual manner using Solvent systems E, F, and G (Table 1). Extracts were co-chromatographed with authentic indole3-propionic acid and indole-3-acrylic acid. The conversion of indole-3-propionic acid to indole-3-acrylic acid was estimated to have proceeded in 30% yield in 72 h. N-carbobenzoxyl-L-tryptophan metabolism in the presence of L-tryptophan Addition of tryptophan to fermentation medium enhanced the ability of

140 --

DEHYDRO'METABOLITE(4.35x 105 M~ -" N - C B Z ' T R Y P T O P H A N (9.67 x 10-5 M )

1.4.

; I N D O L E - 3 - A C R Y L i C ACID(9.,10 x 10"SM)

1-21-0-

w 0.0(9 7 0.6. < cfl 0o4m < 0.20

J

210

2;0 2;0

F i g . 3. U l t r a v i o l e t

spectra

i

2'~0 290 3~0 (Methanol)

,

3~0 ~so 3~0

of Cbz-Trp,

indole-3-acrylic

acid, and the dehydro-metabolite

(II).

C. violaceum cultures to transform Cbz-Trp into its dehydrogenated product. Cultures stored in nutrient broth containing 0.1--0.5 mg/ml L-tryptophan maintained the ability to perform this transformation, while cultures stored only in nutrient broth lost activity upon repeated serial transfers. The influence of medium L-tryptophan on the conversion of Cbz-Trp to the metabolite was evaluated. For this experiment, we selected a culture which had nearly lost the ability to perform the desired conversion due to repeated serial transfer into fresh nutrient broth. Cultures were grown both on the usual medium, and on medium supplemented with the amino acid at a concentration of 0.5 mg/ml. After 24 h, the substrate, Cbz-Trp was added as usual to a final concentration of 1 mg/ml in fermentation media. Samples were evaluated by thin-layer chromatography (Solvent system H). Maximum conversions (100% of substrate utilized) occurred in cultures containing L-tryptophan both in Stage I and Stage II cultures. Conversion yields obtained with cultures containing L-tryptophan only in Stage II cultures were about 70% by thin-layer chromatography estimation. Only traces of the dehydro-metabolite were produced by cultures grown without L-tryptophan. N-Carbobenzoxyl-D-tryptophan; N-trifluoroacetyl-L-tryptophan; N-formyl-L-tryptophan, and N-acetyl-L-tryptophan were also incubated in cells grown with and without L-tryptophan in the medium. All except for N-Cbz-Dtryptophan were metabolized to unidentified products by L-tryptophan-containing C. violaceum cultures. Influence o f L-tryptophan on growth o f C. violaceum Growth curves were obtained with C. violaceum grown in the presence and absence of 0.5 mg/ml L-tryptophan in Stage II culture medium, by a modification of the procedure described by DeMoss and Happel [16]. Culture samples were diluted with an equal volume of ethanol to solubilize particulate pigmented material. To compensate for the greater amount of pigment produced in L-tryptophan-containing cultures, and to obtain a truer representation

141

of growth, the absorbances of 40 000 × g supernatants were deducted from absorbance readings obtained for the whole cell suspensions. Both sets of cultures attained the stationary phase of growth by 24 h, b u t the L-tryptophancontaining culture yielded approximately 35% more growth than the normal cultures.

Resting cell suspension studies C. violaceum cells were harvested from 24-h-old Stage II cultures by centrifugation at 8400 × g for 10 min. The resulting solids were resuspended in 0.1 M phosphate buffer (pH 6.0), and were centrifuged again for 10 min. The sedimented cells were frozen at --20 ° C until required for experimentation.

Influence of incubation conditions, and phenazine methosulfate concentration on the metabolism of Cbz-Trp, and indole-3-propionic acid by resting cells Incubations were conducted with shaking at 180 rev./min, at 27°C, in 10 ml of cells suspended in 0.1 M phosphate buffer (pH 6.0). The suspension represented a 10-fold concenization of cells relative to fermentations. Anaerobic incubations were obtained by purging cell suspensions with N2 for 1 h prior to the addition of substrates. N: was then continuously bubbled through the complete incubation mixtures for the remainder of the experiment. The concentrations of substrates used were: Cbz-Trp, 0.5 mg/ml, 1.5 • 10 -3 M; and indole-3-propionic acid, 0.5 mg/ml, 2.7 • 10 -3 M. Phenazine methosulfate was added to anaerobic incubations to concentrations of 2.6 • 10 -4 ; 1.3 • 10 -4 ; and 2.6 • 10 -s M. 1 h after addition of substrates, samples were taken, extracted and analyzed by thin-layer chromatography (Solvent system E). Aerobically, b o t h Cbz-Trp and indole-3-propionate yielded a b o u t 50% of their respective dehydro-metabolites. Anaerobically, only traces of metabolites were detected. Phenazine methosulfate generally enhanced the conversion, and some dependence on phenazine methosulfate concentration was observed with Cbz-Trp incubations. The reaction observed in the presence of phenazine methosulfate was enzyme mediated, because without cells, no metabolites could be detected in incubation mixtures. Discussion

Structure elucidation of the metabolite was initially confusing because the c o m p o u n d exhibited little indolic character. Comparisons of the ultraviolet spectra of various tryptophan derivatives showed little absorbance in the 280-nm region which is typical for most indoles. In addition, the metabolite produced a b r o w n color when thin-layer chromatography plates were visualized with FeC13 or p-dimethylaminobenzaldehyde-NaNO2 spray reagents. The low resolution mass spectrum indicated an apparent molecular ion of 336 mass units, and the NMR spectrum revealed that the metabolite structure possessed the Cbz-blocking group originally present on the substrate. Hydrogenolytic cleavage of the Cbz group proceeded smoothly, and the major product of the reaction was isolated and identified spectrally and chromatographically as tryptophan. Other reaction products were characterized by thin-layer chromatography as indole-3-pyruvic acid, indole-3-1actic acid, and

142 indole-3-acetic acid. Although the metabolite structure was n o t completely known when the reaction was run, retrospectively, the formation of these products can be rationalized as arising from the dehydro-metabolite II as shown in Fig. 4. Prominent fragment ions in the high-resolution mass spectrum for the metabolite which support the structure as II are represented in Table 2. The assignment of the double bond to the Cbz-Trp sidechain was confirmed by ultraviolet and NMR spectral comparisons of the metabolite with indole3-acrylic acid. Numerous chromatographic systems were employed in the separation of Cbz-Trp from its metabolite in fermentation extracts with little success. Once the structure of the metabolite was clear, thin-layer chromatography systems which separated indole-3-acrylic acid from indole-3-propionic acid [17] were successfully used in isolating and purifying the metabolite. Interestingly, on thin-layer chromatographic plates, indole-3-acrylic acid and the metabolite both gave a brown color with p-dimethylaminobenzaldehyde-NaNO2 spray reagent. Most indoles give blue or purple colors with this reagent. Indole-3-propionic acid was converted into indole-3-acrylic acid by C. violaceum. Other N-blocked-L-tryptophan derivatives were converted to unidentified products by the microorganism. Only N-Cbz-D-tryptophan was not metabolized b y resting cells, or by actively growing cultures of C. violac e u m , indicating some specificity for the biotransformation.

. E...o,-,T,~

H2, Pd/C

0,]

)~ N-CBZ-TRYPTOPHAN (I)

I

-CBZ

L'~

'J.

U

~

-CBZ

TRYPTOPHAN (IV)

Nil 2

COOH

(v,) H

~

~

O

O

H

OOH

H

~ Fig. 4. H y d r o g e n o l y s i s of

[VIII)

COOH

2',3'-dehydro-N-carbobenzoxyltrYptophan.

143

Biological significance o f 2',3'-N-Cbz-dehydrotryptophan (H) a, ~-Unsaturated amino acids have been found as structural components of several peptide antibiotics where they exist in stabilized form through peptide bond linkages [18]. Dehydrotryptophan is a structural component of the antibiotic teliomycin [19]. Gross and coworkers [20,21] related the biological activity of nisin and subtilin directly to the presence of dehydroalanine residues found in both antibiotic structures. The biogenetic mechanism by which dehydro-amino acids are incorporated into such peptides is obscure. The metabolite II could be considered as an adventitiously trapped intermediate of one of the L-tryptophan metabolic pathways of C. violaceum. It is unlikely that such a metabolite would be formed in pyridoxal-mediated reactions, because the formation of the Schiff base intermediate common to these reactions is precluded by the presence of the carbobenzoxyl-blocking group. a, ~-Unsaturated amino acid derivatives have been suggested as intermediates in amino acid oxidase reactions. Dakin [22] described a tautomeric equilibrium between the imino acid VI and the a, ~-unsaturated amino acid V (Fig. 4). Bergmann and coworkers [23] suggested that this intermediate was formed during the conversion of pyruvate to alanine via transamination. Taborsky [24-] provided laboratory evidence for the production of dehydroamino acid intermediates with Ophio-L-amino acid oxidase. With this as a precedent, attempts were made to correlate the biogenesis of II with L-amino acid oxidase enzyme activity. L-Amino acid oxidase from Crotalus adamanteus venom was used according to enzyme assay conditions previously described by Wellner and Meister [25,26]. A number of N-substituted tryptophan derivatives were examined as potential substrates for the enzyme including: N-formyl-tryptophan; N-acetyl-tryptophan; Cbz-Trp; N-trifluoracetyl-tryptophan; and tryptophan itself. Tryptophan was converted into indole-3-pyruvic acid, and indole-3-acetic acid. None of the other tryptophan derivatives were substrates for the enzyme, and no dehydro-metabolite was obtained. Plausible mechanisms for the formation of II include a side-chain dehydrogenase; or side-chain hydroxylase coupled with a dehydratase enzyme system. In the absence of air, resting C. violaceum cells yielded only traces of II or indole-3-acrylic acid from their respective substrates. However, when phenazine methosulfate was added to anaerobic incubation mixtures, yields of dehydrometabolites of Cbz-Trp and indole-3-propionate approached those of controls. These results are suggestive of a dehydrogenase mediated transformation [27]. It is interesting that tryptophan enhances the ability of C. violaceum to perform the biotransformation, and it also prevents the loss of transforming ability when cultures are serially transferred during storage. The mechanism of this enhancement phenomenon is uncertain. It has been shown by others that tryptophan increases growth of C. violaceum [16] a finding which is verified by our results. The increase in growth stimulated by tryptophan, however, is insufficient to account for the significantly higher ability of tryptophan-containing cultures to yield dehydro-metabolites. The metabolic significance of this study in relation to tryptophan metabolism in C. violaceum is unclear. The enhancement of dehydro-metabolite producing activity by tryptophan appears to link the biotransformation to the

144

presence of this amino acid, or a metabolite in fermentation medium. Indole3-pyruvate and indole-3-acetate are tryptophan metabolites in C. violaceum, [3--5] and are formed usually by means of a transaminase [ 4 ] . These same tryptophan metabolites could also form via the action of a side-chain dehydrogenase, albeit by a completely different pathway. Intermediates involved would be similar to V and VI in Fig. 4. Further studies toward understanding this reaction are in progress. The enzyme has been isolated and purified more than 20-fold, and studies relating to further enzyme purification, substrate specificities and enzyme mechanism are being conducted. Acknowledgement P.J.D. and M.G. gratefully acknowledge partial support through the N.S.F., grant GY 10575. References 1 Ballantine,J.A., Barrett, C.B., Beer~ R.J.S., E~Lrdley, S., Robinson, A., Shaw, B.L. and Simpson, T.H. (1958) J. Chem. Soc. 755--760 2 DeMoss, R.D. and Evans, N.R. (1959) J. Bacteriol.78,583--588 3 DeMoss, R.D. and Evans, N.R. (1960) J. Bacteriol.79, 729--733 4 D e Moss, R.D. and Evans, N.R. (1957) Bacteriol Proc. 117 5 Mitoma, C., Weissbach, H. and Udenfriend, S. (1956) Arch. Biochem. Biophys. 63,122--130 6 Mitoma, C., Weissbach, H. and Udenfriend, S. (1955) Nature 175, 994--995 7 Contractor, S.F., Sandler, M. and Wragg, J. (1964) Life Sci. 3,996--1006 8 Bazelon, M., Paine, R.S., Cowie, V.A., Hung, P., Houck, S.C. and Mahanand, P. (1967) Lancet 1, 1130--1133 9 Persson, T. and Roos, B.E., (1968) Nature 217, 854--856 10 Nakazawa, H., Enei, H., Okurmura, S., Yashida, H. and Yamada, H. (1972) F E B S Lett. 25, 43--45 11 Rosazza, J.P., Foss, P., Lemberger, M. and Sih, C.J. (1974) J. Pharm, Sci. 63, 544--547 12 Greenstein, J.P. and Winitz, M. (1961) Chemistry of the A m i n o Acids, Vol. II, p. 938, John Wiley and Sons, N e w York 13 Weygand, F. and Geiger, R. (1956) C h e m . Bet. 89,647---652 14 Greenstein, J.P. and Winitz, M. (1961) Chemistry of the A m i n o Acids, Vol. II, p. 53, John Wiley and Sons, N e w York 15 Kaper, J.M. and Veldstra, M. (1958) Biochim. Biophys. Acta 30, 401--420 16 DeMoss, R.D. and Happel, M.E. (1950) J. Bacteriol. 77, 137--141 17 Hansen, I.L. and Crawford, M.A. (1966) J. Chromatogr. 22, 33{)--335 18 Gross, E. (1970) C R C H a n d b o o k of Biochemistry (Sober, H.A., ed.), 2nd edn p. B-50, C R C Press, Cleveland, Ohio 19 Sheehan, J.C., Mania, P., Nakamttra, S., Stock, J.A. and Maeda, K. (1968) J. A m . C h e m . Soc. 90, 462--470 20 Gross, E.R. and Morell, J.L. (1967) J. A m . C h e m . Soc. 89, 2791--2792 21 Gross, E.R., Morell, J.L. and Craig, L.C. (1969) Proe. Natl. Acad. Sei. U.S. 62,953--956 22 Dakin, M.D. (1926) J. Biol. C h e m . 67,341--350 23 Bergmann, M., Miekeley, A. and Kann, E. (1925) Z. Physiol. C h e m . 146,247--266 24 Taborsky, G. (1955) Yale J. Biol. Med. 127,267--278 25 Wellner, D. and Meister, A. (1960) J. Biol. C h e m . 235, 2013--2018 26 Wellner, D. and Meister, A. (1961) J. Biol. C h e m . 236, 2357--2364 27 Levy, R.H. and Talalay, P. (1959) J. Biol. C h e m . 234, 2014---2021 28 Kirchner, J.G. (1967) Thin Layer Chromatography, p. 53, IntersciencePublishers,Inc., N e w York