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Herbicidal lead compound mimosine and its degradation enzyme

Shinkichi Tawata1, Tran Dang Xuan and Masakazu Fukuta

1 Department of Bioscience and Biotechnology, Faculty of Agriculture,
University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan
Email: b986097@agr.u-ryukyu.ac.jp

Abstract

Leucaena (Leucaena leucocephala de Wit) has great potential as animal feed in the tropics due to high protein content and annual yield. However, the presence of mimosine [β-(N-(3-hydroxy-4-pyridone)-α-amino-propanoic acid] prevents from wide use of leucaena, as its ingestion causes alopecia, growth retardation, cataract, and infertility in animals. Since mimosine can be purified on an industrial scale by use of an ion-exchange resin method, it has been studied as an herbicidal lead compound. We have attempted to utilize mimosine as a profitable and effective herbicidal and antifungal agent. When fungi attack leucaena, mimosine converted to DHP [3-hydroxy-4(1H)-pyridone] by the action of the mimosine degradation enzyme. Thirty-eight unknown and 44 known microorganisms were tested for the catabolization of mimosine and DHP. Escherichia coli E-9 growth was inhibited by mimosine, however, DHP strongly promoted its growth. Aerobacter aerogenes growth was increased by both mimosine and DHP. In another trial, sixteen mimosine derivatives were prepared and tested for herbicidal and antifungal activity. Among these compounds 1-β-cyanoethyl-2- pyridone showed 55.6% inhibition at 100 ppm against Brassica rapa. Moreover, 1-β-carbonxylethyl-4-pyridone showed antifungal activity of 85.3% at 100 ppm against Pythium sp.

Media summary

Mimosine can be utilized as a profitable and effective herbicidal and antifungal agent.

Key Words

Leucaena, mimosine, DHP, herbicidal activity, antifungal activity, degradation enzyme.

Introduction

Mimosine is a free amino acid produced in a large quantity in the leaves, seeds, and other parts of the tree-legume Leucaena (Leucaena leucoephala). It is toxic to animal and therefore animals grazing on Leucaena foliage often show harmful side effects. It can also inhibit the growth and protein synthesis in some microorganisms (Soedarjo et al., 1994). Mimosine inhibits DNA synthesis by preventing the formation of the replication fork and inhibiting deoxyribonucleotide metabolism (Gilbert et al., 1995). This free amino acid is known to chelate metals, bind pyridoxal phosphate, and inhibit the enzymes tyrosine decarboxylase, tyrosinase, and ribonucleotide reductase. We have documented a patented leaching method to leach mimosine from Leucaena so that this legume can be used as food and livestock feed (Tawata, 1990). Since mimosine can be purified on an industrial scale by use of an ion-exchange resin method, its use as a herbicidal lead compound has been investigated. This note illustrates our work to examine the use of mimosine as a profitable and effective herbicidal and antifungal agent.

Methods

Bacterial strains

Forty-four known organisms were used for the catabolization of mimosine and DHP. These bacteria were cultured in a medium containing peptone 0.5%, yeast extract 0.01%, K2HPO4 0.2%, KH2PO4 0.2%, NaCl 0.2%, MgSO4-7H2O 0.01%, and pH of 8.0. For the catabolization test, 0.1% of mimosine or DHP were added into the medium containing yeast extract 0.001% without peptone.

Thirty-eight unknown microorganisms were collected from the leucaena plant growing around Ryukyu University Campus. Five microorganisms were obtained from Leucaena stems, 12 from roots, 13 from top soil, and 8 from deep soil.

Mimosine degradation enzyme

The mimosine degradation enzyme was extracted and purified from young leucaena leaves.

Eighteen amino acids (5 mM) were tested as inhibitors of the enzyme.

Synthesis of mimosine derivatives

4-Hydroxy pyridine (5 mmol) reacted with acrylnitrile (0.08 mmol) for 5 hr at 60oC. The reaction mixture was concentrated and purified by preparative TLC to get 1-β -cyanoethyl-4-pyridone. Sixteen derivatives were similarly prepared by using different reagents as starting materials.

Results and discussion

Bacteria growth and inhibition

Fig 1 shows three known microorganisms having unique characteristics. Escherichia coli Crooks (1222) growth was inhibited by mimosine, but increased by DHP. Aerobacter aerogenes (1232) growth was increased by both mimosine and DHP. Coryne bacterium psudodiphterium (1471) growth was inhibited by DHP, but increased by mimosine. Among the unknown microorganisms, fungus D6-31 growth was inhibited by DHP, but increased by mimosine. Fungus D6-30 growth was inhibited by mimosine, but increased by DHP. Fungus D6-27 growth was dramatically increased by both mimosine and DHP. Fungus D3-6 growth was inhibited by both mimosine and DHP. These four unknown fungi have been selected for future research.

Inhibitors of the mimosine degradation enzyme

The mimosine degradation enzyme was isolated and purified from young leucaena leaves. As the enzyme was only found in the precipitate, it was previously thought to be a membrane protein. The precipitate was treated with Triton X-100, dialyzed, and the enzyme finally was isolated. Eighteen different amino acids (5 mM) were added as inhibitors. The enzyme was inhibited at 47.9% by serine and at 40.2% by tyrosine. The activities of glycine, leucine, isoleucine, and valine were nil as shown in Table 1. Since the enzyme was inhibited by ICH2COOH and p-cloro-mercuribenzoic acid (Fig 2), it was assumed to be a group of SH enzymes.

Table 1. Inhibition of the mimosine degradation enzyme

Amino acid
(1mM)

Relative activity
(%)

Glycine

100

Leucine

100

Isoleucine

100

Valine

100

Asparagine

94.8

Alanine

90.1

Threonine

83.7

Glutamine

82.9

Methionine

76.0

Proline

70.0

Phenylalanine

69.4

Tryptophan

62.5

Histidine

55.5

Aspartic acid

55.0

Glutamic acid

54.9

Cysteine

52.7

Serine

47.9

Tyrosine

40.2

Fig 2. Inhibitors of the mimosine degradation enzyme

Fig 1. Characteristics of known microorganisms affected by mimosine and 3,4-DHP

Fig 3. Effects of the mimosine and FeCl3 combination on growth of Brassica rapa

Synthesis of mimosine derivatives

Although the authors have prepared many types of mimosine derivatives, it has been impossible

to obtain more active herbicidal compounds than mimosine. The growth of Brassica rapa was inhibited by mimosine. Also, different mimosine-FeCl3 quelates (Soedarjo and Borthakur 1998) were preparated and tested for B. rapa growth inhibition with different results. When mimosine-FeCl3 complex (4:6) was tested growth was resumed. It seems that the hydroxyl group on the pyridone ring of mimosine inhibits its herbicidal activity.* Mimosine may exist in different forms in Leucaena. When it is protected by FeC13 or other molecules, mimosine loses the ability of its bioactive properties as shown in Fig. 3.

The authors have discovered that by the presence of mimosine in leucaena, it protects itself from extinction. Mimosine has two potential roles; one is to defend itself from perpetrators. The other is to develop and expand the species. Animals become diseased after ingestion of mimosine. Leucaena creates its own colonies by the release of the allelochemical mimosine.

References

Gilbert D, Neilson A, Miyazawa H, Depamphilis ML, Burhans WC (1995) Mimosine arrests DNA synthesis at replication forks by inhibiting deoxyribonucleotide metabolism. The Journal of Biological Chemistry 270, 9597-9606.

Soedarjo M, Hemscheidt TK, Borthakur D (1994) Mimosine, a toxin present in leguminous trees (Leucaena spp.), induces a mimosine-degrading enzyme activity in some strains of Rhizobium. Applied and Environmental Microbiology 60, 4268-4273.

Soedarjo M and Borthakur D (1998) Mimosine, a toxin produced by the tree-legume Leucaena provides a nodulation competition advantage to mimosine-degrading Rhizobium strains. Soil Biology and Biochemistry 30, 1605-1613.

Tawata S (1990) Effective reduction and extraction of mimosine from Leucaena and the potential for its use as a lead compound of herbicides. In: Casida, J. E (Ed.), Pesticide and Alternatives. Elsevier Science Publishers, Amsterdam, pp. 541-54

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