[156] Ergothioneine degradation (Escherichia coli)

ERGOTHIONEINE D E G R A D A T I O N (E. coli)

[ 156]

105

were also formed when histidine was replaced by certain histidine derivatives and analogs or by several other amino acids. T h e available data indicate the synthesis of /3-alanyl-l-methylhistidine, /3-alanyl-3methylhistidine, fl-alanyl-2-imidazole-3-alanine, /3-alanyl-l,2,4-triazole3-alanine, fl-alanyllysine,/3-alanylornithine, fl-alanylarginine,/3-alanyl-5hydroxylysine, and several other 13-alanyl peptides. T h e enzyme also catalyzes the synthesis of the T-aminobutyryl peptides of histidine, lysine, ornithine, arginine, 1-methylhistidine, and 3-methylhistidine. It is thus evident that the chick pectoral muscle enzyme preparation has a rather broad specificity. Whether a single enzyme is responsible for the synthesis of these various peptides or whether the chick muscle preparation contains more than one enzyme is not yet known. It is of interest that fl-alanyllysine has been found in chick muscle 9 and that T-aminobutyryl histidine is known to occur in brain. TM 9G. D. Kalyankar, unpublished data, (1959); M. Matsuoka, T. Nakajima, and 1. Sano, Biochim. Biophys. Acta, 177, 169 (1969). ,0j. j. Pisano, J. D. Wilson, L. Cohen, D. Abraham, and S. Udenfriend,J. Biol. Chem. 236. 499(1961).

[ 156] E r g o t h i o n e i n e

Degradation

By JOHN HC

C/CH2--CH--COOI+ [

~c~H

~

N

I (CH3)~

SH L- Ergothioneine

(Escherichia coli)

B. WOLFF

H~--C / C H ~

C H - - C OO-

+

N...~c/NH

N(CH~)3

SH 2- Thiolurocanic acid

Trimethylamine

Assay Method Principle. T h e enzymatic activity is determined by measuring the increase in absorbance at 311 m/z caused by the formation of thiolurocanic acid? Ergothioneine has no significant absorbance at this wavelength.

'J. B. Wolff, J. Biol. Chem. 237, 874 (1962).

106

HISTIDINE

[156]

Reagents L-Ergothioneine. Dissolve 26.53 mg in 10 ml o f water. Store at 4 ° or frozen. Before use, this stock solution is diluted 10-fold with buffer to make a 10 -a M solution. Sodium p y r o p h o s p h a t e buffer, or tris(hydroxymethyl)aminom e t h a n e (Tris)-HCl buffer, pH 8.5-9.0, 0.10 M Enzyme

Procedure. In a spectrophotometer cuvette having a 1.0-cm light path are mixed 0.25 ml of 10 -a M ergothioneine solution, 0.1 ml of buffer solution, a n d water to give a final volume of 1.0 ml after addition o f the enzyme preparation. T h e cuvette t e m p e r a t u r e should be 25 °. T h e increase in absorbance at 311 m/z is followed after a d d i n g the enzyme solution. Definition of Enzyme Unit and Specific Activity. One enzyme unit is that a m o u n t which will produce an increase in absorbance at 311 m/z of 0.001 per minute at 25 °. T h e formation of 1 micromole of thiolurocanic acid per minute corresponds to an increase in absorbance of 0.019 per minute. Protein concentration is d e t e r m i n e d by the Lowry method. 2 Specific activity is defined as units per milligram of protein. Application of Assay Method to Crude Extracts. T h e assay m e t h o d described is suitable for use with crude or purified preparations, since no interference is e n c o u n t e r e d at the measuring wavelength. Purification Procedure

Growth. Escherichia coli W is grown on the liquid m e d i u m of Vogel and B o n n e r 3 f r o m which NH4 + has been omitted. Glucose (5 g/liter) serves as the source of carbon, and ergothioneine (5 × 10 -a M) as the sole source o f nitrogen. Flasks containing the inorganic m e d i u m at pH 7.5 are autoclaved, a n d the glucose and ergothioneine solutions are a d d e d aseptically. Cultures are aerated by shaking at 37 ° for 2-4 days. Step 1. Cells are harvested by centrifugation for 10 minutes at 14,000 g and are washed twice with 0.13 M KCI solution. A suspension of washed cells in 0.01 M sodium p y r o p h o s p h a t e buffer, pH 9.0, containing 0.25 M sucrose (5 ml of buffer per g r a m wet weight of cells) is treated for 15 minutes at 4 ° in a Raytheon sonic oscillator (10 kc, 250 W). T h e sonicate is centrifuged for 10 minutes at 14,000 g and the sediment o f cell fragments is discarded. All subsequent steps are carried out at 4 °. *O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R, J. Randall, J. Biol. Chem. 193, 265 (1951); see also E. Layne, Vol. III [73]. SH.J. Vogel and D. M. Bonner,J. Biol.Chem.218, 97 (1956).

ERGOTHIONEINE DEGRADATION (E. coli)

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Step 2. To the supernatant solution granular (NH4)2SO4 (243 g/liter) is added slowly with stirring (40% saturation). The pH of the solution is kept at 9.0 by adding very small volumes of NH4OH. When precipitation is complete, the supernatant fluid is separated by centrifugation and the precipitate is discarded. More (NH4)2SO4 (132 g/liter) is added to bring the solution to 60% of saturation. After standing overnight at 4 ° the precipitate is collected by centrifugation and the supernatant fluid is discarded. The protein is redissolved in a small volume of 10-3 M Tris-HC1 buffer, pH 9.0, and desalted by passage through a column of Sephadex G-50. Step 3. The enzyme solution, brought to pH 9.0 with 10-3M Tris-HCl buffer, is adsorbed on a column (1 cm × 10 cm) of DEAE-cellulose previously equilibrated with 10-2 M Tris-HCl buffer, pH 9.0. The enzyme is eluted with a continuous linear salt gradient. The mixing flask contains 250 ml of 0.01 M Tris buffer, pH 9.0, and the reservoir vessel the same volume of buffer made 1.0 M in KCI. With a flow rate of 1.5 nfi per minute, 5 ml fractions are collected. The 20 tubes containing the highest activity are pooled. The product represents a 2.7-fold purification with 20% recovery of activity. The partial purification procedure is summarized in Table I. TABLE I PARTIAL PURIFICATION OF ERGOTHIONASE FROM Escherichia coli W

Step

Total units

Specificactivity (units/rag protein)

1. Sonicextraction 2. (NH4)2SO4precipitation, 40-60% saturation 3. DEAE-celluloseeluate

62,380

615

30,100 12,800

923 1,650

Properties Stability. The presence of 0.25 M sucrose protects the purified enzyme against inactivation by repeated cycles of freezing and thawing. Stability at 37 ° is greatest at pH 8.5-9.0; at pH 5.0 the half-life of the purified enzyme is reduced to 30 minutes. Other Bacterial Sources. After growth on ergothioneine, the enzyme activity is present in Alcaligenes faecalis, 4 Bacillus subtilis (ATCC 9945), 1 and four strains of Staphylococcus aureus, a Substrate Specificity. Ergothionase is found only in cells grown on ergothioneine. The enzyme is absent from organisms grown on betaine, 4D. Yanasugondha and M. D. Appleman,J. Bacteriol. 74, 381 (1957).

108

HISTIDINE

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choline, hercynine, or histidine. 1 Extracts of cells raised on ergothioneine also show a small amount of histidase s activity, and they degrade thiolurocanic acid. 8 The purified ergothionase does not break down thiolurocanic acid and lacks any trace of histidase activity. 1 pH Optimum. Maximal ergothionase activity occurs at pH 8.5-9.0 in sodium pyrophosphate or Tris buffer. Reversibility. The enzyme does not catalyze the synthesis of ergothioneine from thiolurocanic acid and trimethylamine. 1 Inhibitors. The enzyme is not affected by the presence of 0.1 M glycine or 10 -3 M ethylenediaminetetraacetate, s Cysteine reduces its activity by 32% at concentrations of 0.01 M and higher, at pH 6.9, although glutathione or mercaptoethanol has no effect at similar or higher concentrations, and they do not reverse the inhibition due to cysteine. Iodoacetate reduces ergothionase activity irreversibly to 50% at 7 × 10-4M. Substrate Affnity. The dissociation constant Km for the enzyme-substrate complex, determined by the method of Lineweaver and Burk, 7 is 5.6 x 10-5M. Preparation and Properties of 2-Thiolurocanic acid. The chemical conversion of ergothioneine to thiolurocanic acid has been described by Barger and Ewins. 8 For the enzymatic preparation of the product, 265 mg (1 millimole) of ergothioneine-HCl is incubated in 30 ml of 3.3 × 10 -3 M sodium pyrophosphate buffer, pH 9.0, with 90 mg of E. coli W sonic extract at 37 ° for 4 hours. The pH is reduced to 5 with glacial acetic acid, then the mixture is deproteinized by heating at 100 ° for 5 minutes and filtration through diatomaceous earth. On cooling, yellow crystals of thiolurocanic acid form. The product is recrystallized from aqueous alcohol. It is separated from ergothioneine by chromatography on a 13 x 100 mm Dowex 1-formate column (8% cross-linkage, 100-200 mesh). The solution containing ergothioneine and thiolurocanic acid is applied to a column previously equilibrated with 0. l M sodium formate. Ergothioneine is washed from the resin with water, then thioluroncanic acid is eluted with 1 N formic acid. No melting point is observed for thiolurocanic acid, since decomposition begins at about 265 °. The compound exhibits characteristic optical absorption: the maximum at pH 7 at 311 m g changes reversibly to higher wavelength (up to 340 m/.~) below pH 6 and above pH 9. The molar absorbancy index (pH 7) is 1.9 × 104. Neither ergothioneine nor 5H. Tabor and A. H. Mehler, Vol. II [29]. 6B. Kelly and M. D. Appleman,J. Bacteriol. 81,715 (1961). ~H. Lineweaver and D. Burk,J. Am. Chem. Soc. 56, 658 (1934). 8G. Barger and A.J. Ewins,J. Chem. Soc. 99, 2336 (1911).

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ERGOTHIONEINE DEGRADATION (E. coil)

109

urocanic acid has significant absorbance at 311 m~. When activated at its absorption maximum, thiolurocanic acid exhibits strong fluorescence at 500 m/~. This property makes it possible to detect as little as 10 -2 nanomole of the compound on paper chromatograms (except in the presence of phenol). T h e behavior of thiolurocanic acid and related compounds in several paper chromatographic systems is documented in Table II. The best separations by ionophoresis on filter paper are obtained with 0.1 M sodium phosphate buffer, pH 7.0, sodium barbital buffer, 0.01 M, pH 9.15, or with 0.01 M sodium carbonate (pH 11). TABLE II PAPER CHROMATOGRAPHY OF THIOLUROCANIC ACID AND RELATED COMPOUNDS a

Compound

2-Thiolurocanic acid e-Ergothioneine-HCl 2-Thiolhistidine L-Histidine Urocanic acid Hercynineb

Solvent system (Re) A

B

C

D

0.84 0.25 0.21 0.19 0.62 0.14

0.39 0.45 0.36 0.29 0.32 0.35

0.37 0.95 0.37 0.77 0.71 0.93

0.55 0.39 0.28 0.21 0,75 0.30

aSolvent Systems: (A) 1-Butanol-glacial acetic acid-water (4:1:5 by volume, upper phase); (B) l-butanol-pyridine-water (1:1:1 by volume); (C) phenol-water (HCN vapor) (4:1 by weight); (D) 1-propanol-glacialacetic acid-water (75:1.5: 23.5 by volume). Method: Descending, Whatman No. 1 paper, 25°. Visualization: (1) Ultraviolet light (253.7 m/z)- ergothioneine quenches the fluorescence of the paper, thiolurocanic acid shows bright green fluorescence~ (2) Modified Pauly diazotizing reagent (Melville and Lubschez 1° and TaborH)-ergothioneine first yellow, then mauve; thiolurocanic mauve. (3) Iodoplatinate reagent (Winegard et al.12)-ergothioneine black, hercynine black, others no reaction. bFor preparation see Wolff?

Degradation of Thiolurocanic Acid. A cell-free extract of AIcaligenes faecalis breaks down thiolurocanic acid at pH 8.0 to H2S and glutamic acid .6,9

9j. s. Booth and M. D. Appleman,J. Bacteriol. 85,654 (1963). l°D. B. Melville and R. Lubschez,J. Biol. Chem. 200, 275 (1953). nil. Tabor, Vol. III [90]. 1~H. M. Winegard, G. Toennies, and R.J. Block, Science 108, 506 (1948).

Articles Related to Section II Vol. IV [28]. Methods for Chemical Synthesis, Isolation, and Degradation of Labeled Compounds as Applied in Metabolic Studies of Amino Acids and Proteins. D. M. Greenberg and M. Rotbstein. Vol. V [ 115]. Diaminopimelic Acid Synthesis in Escherichia coil C. Gilvarg. Vol. V [116a]. N-Succinyl-L-diaminopimelic Deacylase. S. H. Kindler. Vol. V [116b]. N-SuccinyI-L-diaminopimelic Glutamic Transaminase. B. Peterkofsky. Vol. V [117]. Diaminopimelic Racemase. E. Work, Vol. V [118]. Diaminopimelic Decarboxylase. E. Work. Vol. VI [92]. c~, e-Diaminopimelic Acid. E. Work.