Investigation of the role of 3-hydroxyanthranilic acid in the degradation of lignin by white-rot fungus Pycnoporus cinnabarinus

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Abstract

An aminophenol, 3-hydroxyanthranilic acid (3-HAA), has been proposed to play important roles in lignin degradation. Production of 3-HAA in Pycnoporus cinnabarinus was completely inhibited by a combination of tryptophan and S-(2-aminophenyl)-L-cysteine S,S-dioxide (APCD) while the fungus grew well and produced high amounts of laccase. The biosynthesis of 3-HAA is mainly through the metabolism of tryptophan in the kynurenine pathway. A minor pathway for 3-HAA synthesis is through the hydroxylation of anthranilic acid during the biosynthesis of tryptophan in the shikimic acid pathway. Through UV irradiation of wild-type P. cinnabarinus (WT-Pc) spores, a 3-HAA-less mutant was produced. Both WT-Pc, under the inhibitory culture condition, and the 3-HAA-less mutant were found to degrade lignin in unbleached kraft pulp as efficiently as the WT-Pc, which unambiguously demonstrated that 3-HAA does not play an important role in the fungal degradation of lignin.

Introduction

White-rot fungi, the only microorganisms able to efficiently degrade lignin, play a very important role in the global carbon cycle. The extracellular phenoloxidases: lignin peroxidase, manganese peroxidase and laccase, secreted by white-rot fungi are the key enzymes in lignin degradation [1]. It has been well documented that laccase alone is not able to degrade non-phenolic lignin structures, which account for 90% of lignin substructures in plant cell walls, unless an organic compound, a laccase-mediator, is present [2]. Since the white-rot fungus, P. cinnabarinus, efficiently degrades lignin while producing only one-isoform of laccase as phenoloxidase [3], [4], 3-hydroxyanthranilic acid (3-HAA), produced by the fungus, has been proposed to be a laccase mediator [5]. It has been demonstrated that a combination of 3-HAA and laccase, purified from P. cinnabarinus, could depolymerize synthetic lignin (DHP) [5]. However, 3-HAA plus laccase cannot delignify unbleached kraft pulp and cannot oxidize 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol, the predominant lignin substructure in wood (unpublished results). Therefore, the exact role of 3-HAA in the fungal degradation of lignin is not yet clear. To clarify this issue, we first need to understand how 3-HAA is produced in the fungus.

3-HAA has been found in bacteria, yeast, fungi, plants and mammals [6], [7]. P. cinnabarinus is, so far, the only white-rot fungus reported to produce 3-HAA. The biosynthesis of 3-HAA in a white-rot fungus has yet to be addressed. 3-HAA is commonly found as an intermediate of the kynurenine pathway, in which tryptophan is first metabolized to kynurenine and finally to NAD [6], [7], [8]. In bacteria, kynureninase (L-kynurenine hydrolase, EC 3.7.1.3) converts kynurenine to anthranilic acid that is subsequently hydroxylated to produce 3-HAA [6]. In animals and plants, L-kynurenine is first converted to 3-hydroxy-L-kynurenine that is subsequently converted to 3-HAA by 3-hydroxykynureninase [6]. In yeast and fungi, both kynureninase and 3-hydroxykynureninase could be present [9]. For example, Neurospora crassa, Aspergillus niger and Penicillium roqueforti produce both kynureninase and 3-hydroxykynureninase, whereas Rhizopus stolonifer produces only 3-hydroxykynureninase [9]. Kynureninase is inducible by tryptophan while 3-hydroxykynureninase is a constitutive enzyme [6]. If 3-HAA derives from the kynurenine pathway in P. cinnabarinus, inhibition of both kynureninase and 3-hydroxykynureninase would block the synthesis of 3-HAA. Since 3-HAA could also derive from the hydroxylation of anthranilic acid [10], and since anthranilic acid is an intermediate during the synthesis of tryptophan in the shikimic acid pathway [11], [12], it is not known if 3-HAA is solely synthesized by the kynurenine pathway in P. cinnabarinus.

In an effort to understand the biosynthesis of 3-HAA and its role in the degradation of lignin, we studied the inhibition of production of 3-HAA in P. cinnabarinus. We found that S-(2-aminophenyl)-L-cysteine S,S-dioxide (APCD), an excellent competitive inhibitor of kynureninase from Pseudomonas fluorescens [13], can inhibit the production of 3-HAA. A combination of tryptophan and APCD can completely block the synthesis of 3-HAA in P. cinnabarinus. Degradation of lignin by P. cinnabarinus under the inhibitory condition has been investigated. To further confirm the role of 3-HAA in the degradation of lignin by the fungus, a mutant that produces laccase but not 3-HAA has been generated via a UV irradiation method and has been used to study the degradation of lignin.

Section snippets

Reagents

L-Tryptophan, 3-hydroxyanthranilic acid (3-HAA), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ethyl acetate were obtained from commercial sources. Cinnabarinic acid (CA) was synthesized from the reaction of 3-HAA (1.0 mM) with the purified laccase (10 U/ml) [14]. S-(2-aminophenyl)-L-cysteine S,S-dioxide (APCD) was synthesized according to an established procedure [13].

Organism

P. cinnabarinus, strain PB (ATCC 204166), was maintained on 2%(wt/vol) malt extract agar plates (M20

Results

It has been demonstrated that laccase rapidly oxidizes 3-HAA to form CA [14]. We found that no 3-HAA was detectable either in the supernatant of the culture or in the EtOAc extract using the spectrofluorometric method. Therefore, the production of 3-HAA by the fungus was indirectly detected by the measurement of CA. A standard sample of CA was prepared from the reaction of 3-HAA with purified laccase and confirmed its identity by comparison of elution time in liquid chromatography. The mass

Discussion

The biosynthetic pathway, shown in Fig. 7 , proposes three possible routes for the synthesis of 3-HAA. The first route would be from anthranilic acid during the synthesis of tryptophan in the shikimic acid pathway. In this route, chorismate is first synthesized from glucose. Anthranilate synthase then converts chorismate to anthranilic acid that subsequently could be hydroxylated to 3-HAA. During the metabolism of tryptophan to NAD, L-tryptophan is first converted to L-kynurenine. From

Acknowledgements

We would like to thank Dr. Thomas E. Johnson in the Department of Chemistry at the University of Georgia for the spectrofluorometric measurement of 3-HAA. We would also like to thank Dr. Dennis Phillips for the detection of CA using LC-MS.

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