Ligninolytic fungi are unique among eukaryotes in their ability to degrade polycyclic aromatic hydrocarbons (PAHs), but the mechanism for this process is unknown. Although certain PAHs are oxidized in vitro by the fungal lignin peroxidases (LiPs) that catalyze lig olysis, it has never been shown that LiPs initiate PAH degradation in vivo. To address these problems, the metabolism of anthracene (AC) and its in vitro oxidation product, 9,10-anthraquinone (AQ), was examined by chromatographic and isotope dilution techniques in Phanerochaete chrysosporium. The fungal oxidation of AC to AQ was rapid, and both AC and AQ were significantly mineralized. Both compounds were cleaved by the fungus to give the same ring-flssion metabolite, phthalic acid, and phthalate production from AQ was shown to occur only under ligninolytic culture conditions. These results show that the major pathway for AC degradation in Phanerochaete proceeds AC -+ AQ --phthalate + CO2 and that it is probably mediated by LiPs and other enzymes of ligninolytic metabolism.Polycyclic aromatic hydrocarbons (PAHs) are major pollutants of both anthropogenic and natural pyrolytic origin, occurring in soils, sediments, and airborne particulates. The crucial step in their biodegradation is oxidative fission of the fused aromatic ring system, an event previously thought unique to certain bacteria (1). Recent evidence necessitates a revision of this view: the lignin-degrading fungi that cause white-rot of wood have also been shown to mineralize a wide variety of aromatic pollutants, including certain PAHs, under culture conditions that promote the expression of ligninolytic metabolism (2-5). A key component of the fungal ligninolytic system is thought to consist of extracellular lignin peroxidases (LiPs), which catalyze the one-electron oxidation of various lignin-related substrates (6-8). LiPs have also been shown to oxidize certain PAHs in vitro, and it has been proposed that they play an important role in the degradation of these pollutants by white-rot fungi (9, 10). However, it has never been demonstrated that LiP-catalyzed oxidation is a significant fate ofany PAH in vivo or that the products of such a reaction are subsequently cleaved to smaller, monocyclic, compounds. In fact, to our knowledge, no PAH ring-fission metabolite other than CO2 has ever been identified in any eukaryote. To address these problems, we have examined the fate of anthracene (AC) in cultures of the ligninolytic basidiomycete Phanerochaete chrysosporium, and we now report that ligninolytic metabolism provides a route for the ring fission of this PAH. AC trans-1,2-dihydrodiol was synthesized from 1,2-dihydroxy-AQ (alizarin) as previously described (12, 13) [ring-'4C]Phthalic acid (12.7 mCi-mmol-1, radiochemical purity >98%) was from Sigma. Unlabeled phthalic acid (99%) and AQ (>98%) were from Kodak. All other reagents were of the highest commercially available quality. MATERIALS AND METHODSTo minimize the artifactual oxidation of AC or its metabolites, all syntheses, cultu...

The ligninolytic fungus Phanerochaete chrysosporium oxidized phenanthrene and phenanthrene-9,10quinone (PQ) at their C-9 and C-10 positions to give a ring-fission product, 2,2'-diphenic acid (DPA), which was identified in chromatographic and isotope dilution experiments. DPA formation from phenanthrene was somewhat greater in low-nitrogen (ligninolytic) cultures than in high-nitrogen (nonligninolytic) cultures and did not occur in uninoculated cultures. The oxidation of PQ to DPA involved both fungal and abiotic mechanisms, was unaffected by the level of nitrogen added, and was significantly faster than the cleavage of phenanthrene to DPA. Phenanthrene-trans-9,10-dihydrodiol, which was previously shown to be the principal phenanthrene metabolite in nonligninolytic P. chrysosporium cultures, was not formed in the ligninolytic cultures employed here. These results suggest that phenanthrene degradation by ligninolytic P. chrysosporium proceeds in order from phenanthrene-PQ-* DPA, involves both ligninolytic and nonligninolytic enzymes, and is not initiated by a classical microsomal cytochrome P-450. The extracellular lignin peroxidases of P. chrysosporium were not able to oxidize phenanthrene in vitro and therefore are also unlikely to catalyze the first step of phenanthrene degradation in vivo. Both phenanthrene and PQ were mineralized to similar extents by the fungus, which supports the intermediacy of PQ in phenanthrene degradation, but both compounds were mineralized significantly less than the structurally related lignin peroxidase substrate pyrene was.

The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.

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