J. Biochem. 85, 785-790 (1979)

Pterin Deaminase from Bacillus megaterium

Purification and Properties

Shinichiro TAKIKAWA, Chizuko KITAYAMA-YOKOKAWA , and Motoo TSUSUE

Biological Laboratory, Kitasato University, Sagamihara, Kanagawa 228

Received for publication, August 24, 1978

A pterin deaminase catalyzing the hydrolytic deamination of various pteridines was found in the bacterium, Bacillus megaterium, and partially purified from bacterial extract. The specific activity was raised 90-fold over that of the crude extract. The pH optimum is around 7.3, and the Km value for 6-carboxypterin is 1.3 mat. The molecular weight of the was estimated by gel filtration to be about 110,000. The enzyme deaminated pterin, 6-carboxy- pterin, biopterin, 6-methylpterin, 7-methylpterin, xanthopterin, 6-hydroxymethylpterin, sepia- pterin, isosepiapterin, folic acid, and 6,7-dimethylpterin to their corresponding lumazines, whereas guanine, 7-carboxypterin, leucopterin, isoxanthopterin, and 6-methylisoxanthopterin did not serve as substrates. The enzyme was inhibited by PCMB and 8-azaguanine.

One of the catabolic pathways for pteridines xanthopterin and isoxanthopterin, but the prop- involves deamination reactions resulting in the erties of the and the bacterial strain were formation of the corresponding lumazines (1). not reported. McNutt (4) also reported an iso- Levenberg and Hayaishi (2) first reported a pterin xanthopterin deaminase in Alcaligenes faecalis. In deaminase [EC 3.5.4.11] in the bacterium, Al- Bombyx mori, two pterin deaminases were found: caligenes metalcaligenes. Levy and McNutt (3) one is sepiapterin deaminase, which is specific for found other bacterial deaminases which deaminate yellow dihydropterins (5); the other is an isoxan- thopterin-specific deaminase (6). The occurrence of mammalian pterin deaminase in rat liver was Abbreviations: The following trivial names are used reported by Rembold and Simmersbach (7). in the text: pterin, 2-amino-4-hydroxypteridine; luma- During the course of studies on the degrada- zine, 2,4-dihydroxypteridine; biopterin, 2-amino-4- tion of sepiapterin (8, 9), a new pterin deaminase hydroxy-6-(L-erythro-1',2'-dihydroxypropyl)pteridine; was found in extracts of Bacillus megaterium. In xanthopterin, 2-amino-4,6-dihydroxypteridine; sepia- the early stages of the investigation, sepiapterin pterin, 2-amino-4-hydroxy-6-lactyl-7,8-dihydropteridine; was used as the enzyme in assays; sub- isosepiapterin, 2-amino-4-hydroxy-6-propionyl-7,8-di- sequently it was replaced by 6-carboxypterin, hydropteridine; leucopterin, 2-amino-4,6,7-trihydroxy- because the latter proved to be a better substrate pteridine; isoxanthopterin, 2-amino-4,7-dihydroxypteri- than sepiapterin. In the present paper, a method dine; folic acid, pteroylmonoglutamic acid. Lumazine for purifying the enzyme from bacterial extracts is derivatives corresponding to sepiapterin, isosepiapterin, described and some properties of the purified and biopterin are referred to as sepialumazine, iso- enzyme are presented. sepialumazine, and biolumazine, respectively.

Vol. 85, No. 3, 1979 785 786 S. TAKIKAWA, C. KITAYAMA-YOKOKAWA, and M. TSUSUE

shows a sharp peak at 360 nm in 0.1 M potassium

MATERIALS AND METHODS phosphate buffer, pH 7.0 (2). The enzyme activity was, therefore, determined spectrophotometrically Bacteria and Culture Conditions-Bacillus by a modification of a previously described method megaterium 63 (10) was kindly supplied by Dr. Irie (13). of the National Institute of Animal Industry.' Cells The standard reaction mixture contained the were cultured at 36°C in brain-heart infusion following components: 6-carboxypterin, 0.25ƒÊmol;

(Eiken) under aerobic conditions. After growth potassium phosphate buffer, pH 7.0, 0.1 mmol, and for 24 h, cells were harvested by centrifugation at enzyme in a final volume of 1.0 ml. The reaction

10,000 x g at 4°C for 15 min, washed by suspending was started by the addition of enzyme. After in a small volume of 0.01 M potassium phosphate incubation at 25°C for an appropriate time, the

buffer, pH 7.0, and collected by centrifugation. reaction was stopped by heating at 100?C for 3 min. Approximately 15 g (wet weight) of cells per liter The cooled reaction mixture was centrifuged when of the medium was obtained. The cells could be necessary. A similarly constituted reaction mix stored at -20°C for at least 3 months without ture containing heat-treated enzyme served as a

appreciable loss of the enzyme activity. control. The decrease in absorbance at 360 nm Chemicals-6-Carboxypterin was prepared by relative to the control solution was measured. alkaline permanganate oxidation of folic acid (11). When 0.25,ƒÊmol of 6-carboxypterin was com-

Sepiapterin and isosepiapterin were prepared by pletely deaminated to 6-carboxylumazine at pH 7.0, the method of Sugiura et al. (12). Sepialumazine the decreased value of the absorbance at 360 nm and isosepialumazine were prepared enzymatically was 1.35. One unit of the enzyme was defined as (13). Biopterin was prepared from bullfrog skin the amount which deaminated 1 nmol of 6-carboxy- (14). Authentic samples of 6-carboxylumazine pterin per 10 min under the standard assay con and 6-hydroxylumazine were generous gifts from ditions. Prof. Matsuura of Nagoya University. Other Ammonia Measurement-For the assay of am pteridines were kindly supplied by Prof. Akino of monia liberation, pteridines were dissolved in 1 Tokyo Metropolitan University. polyvinyl alcohol (6, 20). The reaction mixture P-Sephadex was prepared by the method of contained the following components: pteridine, 2 Peterson and Sober (15), using Sephadex G-25 (fine) ƒÊ mol; potassium phosphate buffer, pH 7.0, 0.2 instead of cellulose powder. Ethoxy-cellulose was mmol; and enzyme in a final volume of 2.0 ml. prepared by the method of Lee and Montgomery After incubation at 25°C for an appropriate time, (16). All other chemicals were obtained from 0.5 ml of I M KHIP04 was added, followed by commercial sources. heating in a boiling water bath for 3 min. Two ml For the calculation of concentrations of pteri- of the deproteinized solution was put into a Conway

dines, the following absorptivities (m-1-cm-1) were micro-diffusion apparatus and ammonia was ana- utilized: 6-carboxypterin, 9.2 x 103 at 365 nm in lyzed by the alkaline-phenol method (21). 0.1 N NaOH (17); 6-carboxylumazine, 8.1 x 103 at

370 nm in 0.1 N NaOH (18). RESULTS Protein Determination-Protein was deter- mined by the method of Lowry et al. (19), using Purification of Pterin Deaminase-All sub- bovine serum albumin as a standard. sequent procedures were carried out at about 4°C Enzyme Activity Assays-The differential spec and all buffers used contained 5 mm 2-mercapto- trum of 6-carboxypterin and 6-carboxylumazine ethanol. Crude Extract-Fifty grams of the frozen bacterial cells was thawed and suspended in 100 nil I The enzyme from B . megaterium IFO 12108 showed of 0.02 M potassium phosphate buffer, pH 7.0, the same substrate specificity and specific activity as that from B. megaterium 63. The latter bacterium gave containing 0.1 mm phenylmethylsulfonyl fluoride. a better yield than the former in the same culture About 50 ml of the suspension was twice sonicated systems, so B. megaterium 63 was chiefly utilized as the for 5 min at 20 KC, 150 W using a Sonore 150 S enzyme source. unit. The sonicated suspension was centrifuged

.J. Biochem. BACTERIAL PTERIN DEAMINASE 787 at 21,000 x g for 30 min and the supernatant fluid 0.01-0.2 M). The flow rate was 24 ml/h and 4-ml was used as a crude extract. fractions were collected. The fractions containing Protamine Treatment-To the crude extract, deaminase activity (Nos. 44-48) were combined 0.1 volume of 2 % protamine sulfate was added and concentrated by ultrafiltration. dropwise with stirring. After stirring for a further The purification procedures and results are 30 min, the precipitate was removed by centrifuga- summarized in Table I. The specific activity of tion at 10.000 x a for 20 min. the final preparation was raised about 90-fold over Ammonium Sulfate Fractionation-Solid am- that of the crude extract. In all subsequent work monium sulfate was added to the supernatant the purified enzyme was used immediately, since solution to a level of 30% saturation. After the the purified enzyme was rather unstable; the activity suspension had been stirred for 30 min, the pre- decreased about 80% after standing overnight at cipitate was removed by centrifugation at 10,000 x g 4°C. The activity was maintained for several for 20 min. Ammonium sulfate was added to the months in a deep freeze (at -20 and -80°C), but supernatant fluid to give 55% saturation. The freezing and thawing caused a severe loss of activity. suspension was stirred for 30 min and the precipi- Determination of Molecular Weight- The mo tate was collected as above. The precipitate was lecular weight of the enzyme was estimated by gel dissolved in a small volume of 0.01 M potassium filtration through a Sephadex G-150 column (1.5 x phosphate buffer, pH 7.0. Ammonium sulfate was 87 cm) equilibrated with 0.1 Mpotassium phosphate removed by gel filtration through a Sephadex G-25 buffer, pH 7.0. Approximately 500 leg of the column (4 x 16 cm) equilibrated with the same enzyme was applied to the column and eluted with buffer. the buffer at a flow rate of 8 ml/h (2 ml fractions). Column Chromatography on DEAF-Cellulose- The following proteins (molecular weights in The eluate from the Sephadex column was applied parentheses) were used as markers: bovine fibrino- to a DEAE-cellulose column (2 x 20 cm) equili- gen (390,000), bovine liver catalase (247,500), brated with 0.01 M potassium phosphate buffer, bovine y-globulin (169,000), bovine serum albumin pH 7.0. After the column had been washed with (67,000), and cytochrome c (12,384). Elution of 100 ml of the buffer, a 400 ml linear gradient of the marker proteins was monitored by determining NaCI (0 to 0.5 M) in buffer was applied to the absorbance at 280 nm, and the enzyme was moni column. The flow rate was 40 ml/h and 5-ml frac- t ored by the spectrophotometric assay of its activity. tions were collected. The active fractions (Nos. The molecular weight of the enzyme was found to 83-88) were combined. he approximately 110,000. Column Chromatography on Hydroxylapatite- -Under the standard assay The combined solution was applied to a hydroxy- conditions, 6-carboxylumazine production in the lapatite column (1.5 x 20 cm) equilibrated with deamination reaction was linear for at least 30 min 0.01 M potassium phosphate buffer, pH 7.0. After and the initial velocity was proportional to the the column had been washed with 40 ml of the enzyme concentration. From the double recipro- buffer, proteins were eluted with a 200 ml linear cal plot of initial velocity versus substrate concen- tration, the Km value for 6-carboxypterin was gradient of potassium phosphate buffer (pH 7.0,

Vol. 85, No. 3, 1979 788 S. TAKIKAWA, C. KITAYAMA-YOKOKAWA, and M. TSUSUE

TABLE II. Effects of inhibitors on pterin deaminase. TABLE III. Comparison of the amounts of ammonia liberated with several pteridines as substrates. The reaction mixture contained the following components:

pteridine, 2 ƒÊmol; potassium phosphate buffer, pH 7.0, 0.2 mmol; enzyme, 23 units, in a final volume of 2.0 ml. Incubations were carried out at 25°C for 4 h. Other assay conditions are described in the text.

calculated to be 1.3 mm, Inhibitors-The inhibitory effects of several compounds on the deaminase activity were studied under the standard assay conditions. As shown in Table II, the enzyme was inhibited considerably by 1 mm 8-azaguanine and p-chloromercuribenzoate (PCMB). The inhibitory effects of fluoride, cya- of ammonia liberated after incubation for 4 h using nide, guanine, and amethopterin were slight or nil several pteridines as substrates. From the results, at the concentrations tested. pterin, 6-carboxypterin, biopterin, and 6-methyl- Ammonia Liberation during the Reaction- pterin are better substrates than the other pteridines Ammonia production increased linearly with time examined. for at least 2 h when 2 ƒÊmol of 6-carboxypterin The effect of pH on the deaminase reaction was incubated with 35 units of the enzyme at 25°C was examined by determining ammonia production. in 0.1 M potassium phosphate buffer, pH 7.0. As shown in Fig. 1, the optimum pH is around 7.3.

Under the above conditions, 0.24 ƒÊmol ammonia It was found that the enzyme activity was greatly and 0.21 ƒÊmol 6-carboxylumazine were produced decreased when Tris was used as a buffer. after incubation for 30 min. Therefore, the ratio Isolation of the Reaction -About of the amount of ammonia liberated to that of 10 mg of 6-carboxypterin was incubated overnight 6-carboxylumazine formed was about 1 : 1. with 400 units of the enzyme at 25°C in 0.1 M When 2 ƒÊmol each of 6-carboxypterin, pterin, potassium phosphate buffer, pH 7.0. The reaction and biopterin were incubated overnight under the product was purified by successive chromatography conditions described above, 2 ƒÊmol of ammonia on a P-Sephadex column (5 x 30 cm), an ethoxy- was formed in each case and a new single spot cellulose column (5 x 30 cm), and a Sephadex appeared on a paper chromatogram instead of the column (G-25, fine, 5 x 30 cm). The product was substrate. On the contrary, no ammonia was eluted with distilled water from these columns. detected when guanine, 7-carboxypterin, leuco- The ultraviolet absorption spectra of the substance pterin, isoxanthopterin, and 6-methylisoxantho- were identical with those of authentic 6-carboxy- pterin were used as substrates under the same lumazine. The paper chromatographic and elec conditions. These findings, together with the trophoretic properties were also the same as those results obtained by paper chromatographic analysis, of 6-carboxylumazine. indicate that the purified enzyme is not contami- Biolumazine was also isolated by using bio nated with guanase and that the latter pterins do pterin as a substrate. The ultraviolet absorption not serve as substrates. spectra of the purified product showed peaks at Table III shows a comparison of the amounts. 218, 257, and 370 nm in 0.1 N NaOH, and at 230

J. Biochem. BACTERIAL PTERIN DEAMINASE 789

of ammonia, together with the observation that the deamination reaction also takes place under anaero- bic conditions, the reaction can be formulated as follows:

6-carboxypterin+H20 --> 6-carboxylumazine +NH3 One of the characteristic properties of the present enzyme is its low specificity for pterin sub strates: it can deaminate a variety of pterins , in cluding dihydropterins such as sepiapterin and isosepiapterin. In this respect, this enzyme resem bles A. metalcaligenes deaminase (2). However, the former deaminates xanthopterin and 6,7- dimethylpterin and is scarcely inhibited by KF, while the latter does not deaminate these pterins and is inhibited by KF. Another enzyme which is capable of deaminating sepiapterin is the deaminase from B. mori (5), but the enzyme prefers sepia-

Fig. 1. Effect of pH on the deaminase activity. The pterin specifically as a substrate and utilizes other reaction mixture contained the following components: 6-acyl-7,8-dihydropterins to a lesser extent (23);

6-carboxypterin, 2 ƒÊmol; enzyme, 28 units; buffer, 0.2 furthermore it is susceptible to KF (24), as is the mmol, in a final volume of 2.0 ml. The buffers were deaminase from A. metalcaligenes. potassium phosphate (0) or Tris-HCI (0) at various Enzyme instability and a pH optimum near pH values. Incubations were carried out at 25°C for 6.5 are features that are common to the previously 60 min. Other assay conditions are described in the reported deaminases (2, 6, 7) except for sepiapterin text. deaminase. Sepiapterin deaminase is very stable and has a pH optimum at 8.0 (13). Pterin deami and 330 nm in 0.1 N HCI. The spectral character- nase in B. megaterium, presented in this paper, is istics resemble those of biopterin (22). However, rather unstable and has a pH optimum at 7.3, when the product was oxidized with alkaline properties which place it midway between the other permanganate, the resulting compound was identi- types of deaminases. cal with 6-carboxylumazine in both ultraviolet The reported occurrence of unconjugated absorption spectra and paper chromatographic pterins in bacteria indicates that they must be behavior. present in low concentrations. Therefore the In addition to 6-carboxypterin and biopterin, amount of pterins in bacteria may well be regulated the following pteridines were shown to be sub- by the deamination reaction. However, elucida strates for the enzyme by paper chromatographic tion of the exact biological role of pterin deaminase and electrophoretic analyses: pterin, xanthopterin, in bacteria must await further studies. sepiapterin, isosepiapterin, 6-methylpterin, 6- hydroxymethylpterin, 6,7-dimethylpterin, and 7- The authors wish to express their gratitude to Prof. M. Akino of Tokyo Metropolitan University and to Dr. methylpterin. W.L. Gyure of North Central Bronx Hospital for critical readings of the manuscript. DISCUSSION

REFERENCES By using 6-carboxypterin and biopterin as substrates for this newly found pterin deaminase, the corre- 1. Rembold, H. & Gyure, W.L. (1972) Angew. Chem. sponding lumazines were isolated from the reaction Internat. Edit. 11, 1061-1072 mixture and identified. From balance studies on 2. Levenberg, B. & Hayaishi, 0. (1959) J. Biol. Chem. the formation of 6-carboxylumazine and liberation 234,955-961

Vol. 85, No. 3, 1979 790 S. TAKIKAWA, C. KITAYAMA-YOKOKAWA, and M. TSUSUE

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J. Biochem.