Biochimica et Biophysica Acta, 743 (1983) 431-436 431 Elsevier Biomedical Press

BBA 31563

O X I D A T I O N OF ACETYLPOLYAMINES BY EXTRACELLULAR P O L Y A M I N E OXIDASE P R O D U C E D BY P E N I C I L L I U M sp. No. PO-1

YOSHINORI KOBAYASHI a TOSHIHIKO HIGASHI b HARUO MACHIDA b, SHINJIRO IWASAKI b and KOKI HORIKOSHI a a Laboratory of Bacteriology and Ecology, The Institute of Physical and Chemical Research, Wako, Saitama 351, and b Tokyo Research Laboratory, Meito Sangyo Co. Ltd., Ishikawacho 2973, Hachioji, Tokyo 192 (Japan)

(Received September 9th, 1982)

Key words: Acetylpolyamine oxidation; Polyamine oxidase; (Penicillium)

The oxidation of acetylpolyamines by an extracellular polyamine oxidase of Penicillium sp. No. PO-1 was investigated. The optimal pH value for oxidation of acetylpolyamines was 6.0. The purified enzyme oxidized spermidine, spermine, N t-acetyispermidine, N S-acetylspermidine, N I°S-diacetylspermidine, N J-acetylsper- mine and N la2-diacetylspermine. The relative velocities for oxidation of acetylpolyamines were lower than those of spermidine and spermine. The K m values for oxidation of acetylpolyamines were higher than those of spernfi'dine and spermine. The enzyme split N Lacetylspermidine and NS-acetylspermidine at the same position of the linkage as in spermidine oxidation. N t-Acetyispermine was changed to N I-acetyispermidine. This oxidation mechanism was different from that of rat liver polyamine oxidase. N t-Acetylspermine inhibited the oxidation of spermine. Putrescine, NS-acetylspermidine and N t't2-diacetylspermine also inhibited the N I-acetylspermidine oxidation by the enzyme.

Introduction

A previous paper of ours [1] described the pro- duction of an extracellular polyamine oxidase by Penicilliurn sp. No. PO-1 in medium containing 1,3-propanediamine as an inducer. The enzyme was purified by a chromatofocusing method and the properties were investigated in detail [2].

Recently, it has been reported that polyamine oxidase purified from rat liver oxidized several kinds of acetylpolyamines [3,4].

We also found that polyamine oxidase of Peni-

cilliurn sp. No. PO-1 could oxidize acetylpoly- amines. In this paper, substrate specificity of the enzyme and oxidation mechanisms of these sub- strates will be discussed.

Materials and Methods

Polyarnine oxidase. The enzyme was purified from the culture filtrates of Penicilliurn sp. No.

PO-1 by the method described previously [2]. The preparation was homogeneous on SDS-poly- acrylamide gel electrophoresis. The specific activ- ity was 13 050 un i t s /mg protein.

Enzyme assay. Polyamine oxidase activity was assayed spectrophotometrically by measuring the amount of quinoneimine dye, based on the forma- tion of H 2 0 2. The assay system contained 0.2 ml of 500 mM sodium acetate buffer (pH 4.0), 0.2 ml of 0.2% (v /v ) N,N-dimethylaniline solution, 0.1 ml of 0.2% (w/v) 4-aminoantipyrine solution, 0.1 ml of peroxidase solution (90 units/ml) , 0.1 ml of 10 mM spermine tetrahydrochloride solution and 0.3 ml of the enzyme solution of appropriate dilu- tion in a cuvette with a light path of 1 cm. The increase in the absorbance at 565 nm was followed with a Hitachi spectrophotometer model 200-10 at 30°C. 1 unit of the enzyme activity was defined as the amount of the enzyme which formed l nmol of H202 per min under the conditions described

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above. When the assay was carried out at pH 6.0, 0.2 ml of 500 mM phosphate buffer (pH 6.0) was used instead of 500 mM sodium acetate buffer (pH 4.0) mentioned above.

Determination of 11:02. The amounts of H202 formed from N I-acetylspermidine or N l-acetyl- spermine by the oxidation with polyamine oxidase was measured by a method analogous to the en- zyme assay mentioned above.

Determination of putrescine. The amount of putrescine formed from N]-acetylspermidine or Nl-acetylspermine by the polyamine oxidase was measured in an amino acid analyzer [2].

Thin-layer chromatography. The products from acetylpolyamines by the enzyme action were analyzed by thin-layer chromatography using Funagel (Funakoshi Co., Ltd., Tokyo). The solvent system used was chloroform/methanol/am- monium hydroxide (2:2: 1) and polyamines were detected by ninhydrin staining.

Chemicals. Spermidine trihydrochloride and spermine tetrahydrochloride were obtained from Nakarai Chemicals Co., Ltd.. Putrescine dihydro- chloride and ca.daverine dihydrochloride were purchased from Wako Pure Chemical Industries, Ltd. N-Acetylspermidine, N 8-acetylspermidine and N ~-acetylputrescine were prepared as described by Tabor et al. [5]. Nl-Acetylcadaverine and N]'8-di- acetylspermidine were prepared by a method anal- ogous to that described by Tabor et al. [5]. These acetylpolyamines were purified by chromatogra- phy on a Amberlite CG-50 according to the method of Tsuji et al. [6]. N|-Acetylspermine and NL]2-di- acetylspermine were kind gifts from Dr. N. Seiler. The purity and identity of these acetylpolyamines were confirmed by thin-layer chromatography.

R e s u l t s

Effect of pH for the oxidation of acetylpolya- mines. It was found that polyamine oxidase pro- duced by Penicillium sp. No. PO-1 oxidized acetylpolyamines. The optimal pH for the oxida- tion of spermidine and spermine was around 4.0 [2]. However, when Nl-acetylspermidine, N 8- acetylspermidine and N Lacetylspermine were used as substrates, the optimal pH was 6.0 (Fig. 1).

Substrate specificity and kinetic constants for acetylpolyamines. The substrate specificities for

100"

8O"

._) < ._~ 4 0

'~ 2O

I I I 4 5 6 7 8 9

pl-!

Fig. 1. Optimal pH values for acetylpolyamines. The enzyme activity was assayed using three kinds of acetylpolyamines (1 mM) as substrates. The buffer systems used were as follows: pH 3.5-6,100 mM sodium acetate buffer; pH 6-8, 100 mM phosphate buffer; pH 8-9, 100 mM Tris-HCl buffer. (O), N]-acetylspermidine, (e), NS-acetylspermidine, (n), Nl.acetyl- spermine.

several kinds of acetylpolyamines were investi- gated under the standard assay conditions at pH 6.0. As shown in Table I, the enzyme oxidized

TABLE I

RELATIVE VELOCITIES AND KINETIC CONSTANTS FOR POLYAMINES AND ACETYLPOLYAMINES

The assay was carried out using standard assay method at pH 6.0. The velocities for various kinds of substrates were related to that of spermidine as 100%. 100% of relative rate was equivalent to 7.69 mmol oxidized/min per mg protein. The enzyme used was 45 units, n.d., not determined.

Substrate Rela- K m Vma x rive (FM) (/~mol rate H 202 / mg

protein per min)

Spermidine 100 Spermine 76 N ]-Acetylspermidine 44 N S-Acetylsperrnidine 25 N l'S-Diacetylspermidine 0.3 N ]_Acetylspermine 14 N l, 12.Diacetyispermin e 4.3 Putrescine 0 Cadaverine 0 N ]-Acetylputrescine 0 N LAcetylcadaverine 0

0.5 10.3 0.2 7.4

32.3 3.1 26.3 1.4 n.d, n.d.

1.6 5.4 6.4 1.5

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N~-acetylspermidine, N8-acetylspermidine, NLa-diacetylspermidine, NLacetylspermine and Nl:2-diacetylspermine. The relative velocities for the oxidation of acetylpolyarnines were lower than those for spermidine and spermine.

K m and Vma X values for these substrates were estimated by Lineweaver-Burk plots (Table I). K m

values for spermidine and spermine were smaller and Vma X values for them were larger than values for acetylpolyamines.

Oxidation mechanisms. As reported in a previ- ous paper [2], spermine and spermidine were oxidized as follows:

The reaction products of four kinds of acetyl- polyamines were analyzed by TLC.

NLAcetylspermidine and NS-acetylspermidine. As shown in Fig. 2, putrescine and N I- acetylputrescine were formed from NLacetyls- permidine and N S-acetylspermidine, respectively. These results show that the cleavage points of these acetylspermidines by the enzyme action are

3 4 the same ( - C - N - ) as in the case of spermidine

oxidation. The stoichiometry of the reaction was examined

1 2 3 4 5 6 7 8 9 10 11 12 N H 2 _ C H 2 - C H 2 - C H 2 - N H - C H 2 - C H 2 - C H 2 - C H 2 - N H - C H 2 - C H 2 - C H 2 -NH2 + H20 + 02 spermine

N H 2 _ C H 2 - C H E - C H 2 - N H - C H 2 - C H 2 - C H E - C H 2 - N H 2 + C H O - C H 2 - C H 2 - N H 2 + H202 spermidine 3-aminopropion

aldehyde

2 0 + 0 2

N H 2 - C H 2 - C H 2 - C H O + N H 2 - C H 2 - C H 2 - C H 2 - C H 2 - N H 2 +H202 putrescine

as follows: 1 #mol of Nl-acetylspermidine was oxidized by 100 units of the enzyme in 0.5 ml of 10 mM phosphate buffer (pH 6.0) at 30°C for 1 h, and 967 nmol of H202 were detected. Also, putrescine formed was 945 nmol.

Fig. 2. Analysis of the reaction products of acetylspermidines. 1 /~mol of NLacetylspermidine (A) or NS-acetylspermidine (B) was oxidized by 100 units of the enzyme in 10 mM phosphate buffer (pH 6.0). Put, putrescine; Spd, spermidine.

TABLE I1

INHIBITION OF SPERMINE OXIDATION

The assay was carried out under the standard assay conditions at pH 6.0 using 5 #M of spermine as a substrate.

Substance Concentration Rela- (/~ M) tive

rate

None 100 Putrescine 1000 100 N i.Acetylputrescine 1000 1 O0 N LAcetylspermidine 100 100

1000 88 N S-Acetylspermidine 100 96

1000 62 N t.Acetylspermine 10 45

100 11 N 1,12_Diacetylspermin e 100 85

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Based on the results mentioned above, the oxidation pathways of N I-acetylspermidine and N S-acetylspermidine were summarized as follows:

oxidation by Penicillium enzyme were also in- vestigated. Spermine, spermidine and NLacetyl- putrescine showed no inhibition. However,

N Lacetylspermidine 1 2 3 4 5 6 7 8

CH 3 C O - N H - C H 2 - C H 2 - C H 2 - N H - C H 2 - C H 2 - C H 2 - C H 2 - N H 2 + H 2 0 + 02

C H 3 C O - N H - C H 2 - C H 2 - C H O + NH 2 - C H 2 - C H 2 - C H 2 - C H 2 - N H 2 + H202 putrescine

N 8-acetylspermidine

1 2 3 4 5 6 7 8 N H 2 - C H 2 - C H 2 - C H 2 - N H - C H 2 - C H 2 - CH 2 - CH 2 - N H - C O C H 3 + H 2 0 + 02

N H 2 - C H 2 - C H 2 - C H O + NH z - CH 2 - C H 2 - C H 2 - C H 2 - N H - C O C H 3 + H202 N Lacetylputrescine

NLAcetylspermine and Nt'12-diacetylspermine. When N Lacetylspermine was used as a substrate, a spot of N I-acetylspermidine appeared first at an early period, and then after a long incubation a spot of putrescine was detected (Fig. 3A). These

9 10 results show the enzyme split - N - C - . The

amounts of H202 and putrescine formed from 1 #mol of NLacetylspermine were 1.87 gmol and 973 nmol, respectively. The oxidation of N l- acetylspermine was summarized as follows:

putrescine, NS-acetylspermidine and N l'12-di- acetylspermine inhibited the activity. The types of inhibition and K i values were determined from Lineweaver-Burk plots at pH 6.0: K i putrescine, 55.7 #M (competitive); K i NS-acetylspermidine, 11.2/~M (competitive); K i Nl:2-diacetylspermine, 173 #M (non-competitive).

N t-acetylspermine

1 2 3 4 5 6 7 8 9 10 11 12

CH 3CO-CH 2 - C H 2 - C H 2 - N H - C H 2 - C H 2 - C H 2 - C H 2 - N H - C H 2 - C H 2 - C H 2 - N H 2 + H 2 0 + O 2 $

C H 2 C O - C H 2 - C H 2 - C H 2 - N H - C H 2 - C H 2 - C H 2 - C H 2 - N H 2 + C H O - C H 2 - C H 2 - N H 2 + H 2 0 N J-acetylspermidine

N I-Acetylspermidine was further oxidized to putrescine as mentioned above. In the case of N 1.12_diacetylspermine, N Lacetylspermidine was formed first, and then putrescine appeared (Fig. 3B). This result shows that the enzyme splits

3 4 9 10 - C - N - o r - N - C - .

Inhibition. The inhibitory effects of putrescine and acetylpolyamines on the oxidation of sper- mine was investigated. As shown in Table II, N l-acetylspermine strongly inhibited spermine oxidation. The inhibitory effects of polyamines and acetylpolyamines on N I-acetylspermidine

Discussion

The metabolism of polyamines has been studied extensively in mammalian cells. Recent studies showed that the conversion of spermidine into putrescine in rat liver and kidney was catalized by the sequential action of spermidine-Nl- acetyltransferase activity and polyamine oxidase activity, and that the conversion was regulated by the amount of spermidine-NI-acetyltransferase ac- tivity [3,4,7-15].

Bolkenius and Seiler [3] reported the oxidation of acetylpolyamines in rat liver. They purified

Fig. 3. Analysis of the reaction products of acetylspermines. 1 #mol of NI-acetylspei'mine (A) or Nl'12-diacetylspermine (B) was oxidized by 1000 units of the enzyme in 10 mM phosphate buffer (pH 6.0). Put, putrescine; Spd, spermidine; Spm, sper- mine.

polyamine oxidase from rat liver and showed that the enzyme oxidized N ~-acetylspermine, N 1.12_di_ acetylspermine and Nl-acetylspermidine, in addi- tion to spermine. Based on K m and Vma x values for these substrates, they postulated that catabolic pathways exist whereby spermine and spermidine are oxidized via N I-acetylspermine and N l'12-di- acetylspermine, and N l-acetylspermidine, respec- tively. However, NS-acetylspermidine, which was not a substrate of polyamine oxidase, was deacetylated by acetylspermidine deacetylase, as reported by Libby [15], and spermidine occurring was oxidized as described above.

Recently, Matsui et al. [16] described the occur- rence of spermidine-Nl-acetyltransferase activity in Escherichia coli. However, as far as we know, there are no reports on the oxidation system of acetylpolyamines in microorganisms.

Polyamine oxidase purified from the culture

435

filtrates of Peniciilium sp. No. PO-1 oxidized spermidine and spermine [2] by the same mecha- nism as reported for the enzymes produced by Penicillium chrysogenum [17], Aspergillus terreus [18] and Pseudomonas aeruginosa [19].

It was found that the enzyme also oxidized several kinds of acetylpolyamines. There were dif- ferent substrate specificities for N8-acetylspermi- dine from Penicillium sp. No. PO-1 enzyme and from rat liver.

From the results o n g m and Vma x values of Penicillium enzyme for the substrates, it was clear that spermidine and spermine were more suitable substrates for the enzyme than were acetylpolya- mines. Although no crucial results have been ob- tained, it is highly possible that the direct oxida- tion pathway, spermine being oxidized to putres- cine via spermidine, is dominant compared to the pathway via acetylpolyamines.

Rat liver enzyme oxidized N~-acetylspermine and yielded spermidine [3]. Suzuki et al. [4] re- ported that the acetamide group - C 3 - N - C 4 chain was required as a substrate for rat liver enzyme. However, Penicillium enzyme oxidized N 1- acetylspermine and yielded N l-acetylspermidine. This suggests that the N H 2 - C 3 - N - C 4 chain is split by the enzyme in preference to the acetamide group - C 3 - N - C 4 chain. This result supports spermidine and spermine being more suitable sub- strates than acetylpolyamines for the enzyme men- tioned above.

N I-Acetylspermine inhibited spermine oxida- tion by Penicillium enzyme. This may suggest that there is a control system between the two kinds of catabolic pathways mentioned above. It has been reported [3] that N J-acetylspermidine oxidation by rat liver enzyme was inhibited by spermine, spermidine and N S-acetylspermidine. Therefore, there were different inhibition properties for N ~- acetylspermidine oxidation between Penicillium enzyme and that of rat liver.

Acknowledgement

The authors are very grateful to Dr. N. Seiler, Centre de Recherche Merrell International, Stras- bourg, France, for providing us with the acetyl- polyamines.

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