Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Biotransformation of biphenyl by the filamentous fungus <Emphasis Type="Italic">Talaromyces helicus</Emphasis>

The filamentous fungusTalaromyces helicus , isolated from oil-contaminated sludge, oxidizes biphenyl via 4-hydroxybiphenyl to the dihydroxylated derivatives 4,4-dihydroxybiphenyl and 3,4-dihydroxybiphenyl, which, to a certain extent, are converted to glycosyl conjugates. The sugar moiety of the conjugate formed from 4,4-dihydroxybiphenyl was identified as glucose. Further metabolites: 2-hydroxybiphenyl, 2,5-dihydroxylated biphenyl, and the ring cleavage product 4-phenyl-2-pyrone-6-carboxylic acid accumulated only in traces. From these results the main pathway for biotransformation of biphenyl in T. helicus could be proposed to be the excretion of dihydroxylated derivatives (>75%) and their glucosyl conjugates (<25%).     

A novel reaction: meta hydroxylation of biphenyl by an actinomycete

Several yeasts and actinomycetes were examined for their ability to produce 4,4′-dihydroxybiphenyl via biphenyl hydroxylation. Although none was found capable of producing the desired product, one of the actinomycetes produced the meta hydroxylation product, 3-hydroxybiphenyl, exclusively.     

Biotransformation of biphenyl by Paecilomyces lilacinus and characterization of ring cleavage products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Biodegradation of biphenyl by the ascomycetous yeast Debaryomyces vanrijiae

Cells of the yeast strain Debaryomyces vanrijiae SBUG 770, grown with glucose, converted biphenyl to 4-hydroxybiphenyl as the major metabolite. In addition, 2-hydroxybiphenyl was formed in minor amounts. No further degradation of these substances was detected. However, these monohydroxylated derivatives were oxidised by alkane-grown cells in the presence of the co-metabolic substrate, tetradecane. Under these conditions 2-hydroxybiphenyl was oxidised to 2,5-dihydroxybiphenyl, and 4-hydroxybiphenyl was rapidly metabolised by formation of two major metabolites. One was identified as 3,4-dihydroxybiphenyl. Characterisation of the second product as 4-phenylmuconolactone points to a further metabolism of the initially formed dihydroxylated biphenyl via ortho-ring fission. Received: 8 May 1998 / Received revision: 26 June 1998 / Accepted: 2 July 1998     

Biotransformation of Biphenyl by Paecilomyces lilacinus and Characterization of Ring Cleavage Products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4′-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases

In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.     

Biotransformation of biarylic compounds by yeasts of the genus trichosporon

The biotransformation of biphenyl, dibenzofuran, and diphenyl ether by 24 strains belonging to 18 species of the genus Trichosporon was investigated to assess the taxonomic relevance of this property at species and genus level. With the exceptions of T. brassicae and T. porosum CBS 2040, all other strains were able to transform the parent compounds to monohydroxylated intermediates. A second hydroxylation on the same aromatic ring was carried out by fewer strains and depended on the substrate. It appears that this step is the rate-limiting one in the biotransformation of the biarylic compounds tested. Ring fission of dihydroxylated derivatives of biphenyl was observed within 12 species. The aromatic ring system of dihydroxylated dibenzofuran was cleaved by strains of 5 species, while strains of 13 species were able to cleave the aromatic ring system of dihydroxylated diphenyl ether. Only 4 strains out of 18 species were able to cleave the aromatic ring system of all three parent compounds. These most active yeasts belong to the species T. coremiiforme, T. montevideense, T. mucoides, and T. sporotrichoides. In addition, strains of the species Cryptococcus curvatus and Cryptococcus humicola, closely related to the genus Trichosporon, were tested in parallel.     

Novel insights into the fungal oxidation of monoaromatic and biarylic environmental pollutants by characterization of two new ring cleavage enzymes

The phenol-degrading yeast Trichosporon mucoides can oxidize and detoxify biarylic environmental pollutants such as dibenzofuran, diphenyl ether and biphenyl by ring cleavage. The degradation pathways are well investigated, but the enzymes involved are not. The high similarity of hydroxylated biphenyl derivatives and phenol raised the question if the enzymes of the phenol degradation are involved in ring cleavage or whether specific enzymes are necessary. Purification of enzymes from T. mucoides with catechol cleavage activity demonstrated the existence of three different enzymes: a classical catechol-1,2-dioxygenase (CDO), not able to cleave the aromatic ring system of 3,4-dihydroxybiphenyl, and two novel enzymes with a high affinity towards 3,4-dihydroxybiphenyl. The comparison of the biochemical characteristics and mass spectrometric sequence data of these three enzymes demonstrated that they have different substrate specificities. CDO catalyzes the ortho-cleavage of dihydroxylated monoaromatic compounds, while the two novel enzymes carry out a similar reaction on biphenyl derivatives. The ring fission of 3,4-dihydroxybiphenyl by the purified enzymes results in the formation of (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid. These results suggest that the ring cleavage enzymes catalyzing phenol degradation are not involved in the ring cleavage of biarylic compounds by this yeast, although some intermediates of the phenol metabolism may function as inducers.     

Hydroxylation of biphenyl by Aspergillus parasiticus: Approaches to yield improvment in fermenter cultures

We carried out experiments designed to increase the rate of production of 4,4'-dihydroxybiphenyl (biphenol) from biphenyl by Aspergillus parasiticus. We show that 0.5 mg/ml biphenyl, the substrate for the reaction, significantly inhibits growth of the organism and that at 0.04 mg/ml, 2-hydroxybiphenyl or 4-hydroxybiphenyl (an intermediate of the reaction) strongly inhibit oxygen uptake, probably by inhibition of mitochondrial electron transport. Both factors may contribute to the low hydroxylation rates observed previously [J. H. Golbeck and J. C. Cox, Biotechnol. Bioeng., 26, 434 (1984)]. We therefore adapted the organism to the presence of 0.08 mg/ml 2- and 4-hydroxybiphenyl in the growth medium and found that cultures of adapted strains hydroxylated biphenyl at rates ca. three-fold faster than control cultures. Once the fungal mycelia were grown, they could be recycled at least twice into fresh fermentation broth. Recycled organisms were capable of hydroxylating biphenyl more rapidly than cells in the primary fermentation culture and there was no lag period between introduction of biphenyl and the onset of hydroxylation. Cell recycle thus results in a considerable saving in carbon costs and fermentation time.     

Novel Ring Cleavage Products in the Biotransformation of Biphenyl by the Yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4′-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-Trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Oxidation of biphenyl by the Cyanobacterium,Oscillatoria sp., strain JCM

The oxidation of biphenyl by Cyanobacterium, Oscillatoria sp., strain JCM was studied. The organism grown photoautotrophically in the presence of biphenyl oxidized biphenyl to form 4-hydroxybiphenyl. The structure of the metabolite was elucidated by ultraviolet and mass spectra and shown to be identical to authentic 4-hydroxybiphenyl. In addition this metabolite had properties indentical to 4-hydroxybiphenyl when analyzed by thin-layer and high-pressure liquid chromatography. Experiments with [¹⁴C]-biphenyl showed that over a 24 h period the organism oxidized 2.9% of the added biphenyl to ethyl acetate-soluble products.Abbreviations tlc thin-layer chromatography - hplc high pressure liquid chromatography     

Metabolism of 2,2′- and 3,3′-Dihydroxybiphenyl by the Biphenyl Catabolic Pathway of Comamonas testosteroni B-356

The purpose of this investigation was to examine the capacity of the biphenyl catabolic enzymes of Comamonas testosteroni B-356 to metabolize dihydroxybiphenyls symmetrically substituted on both rings. Data show that 3,3′-dihydroxybiphenyl is by far the preferred substrate for strain B-356. However, the dihydrodiol metabolite is very unstable and readily tautomerizes to a dead-end metabolite or is dehydroxylated by elimination of water. The tautomerization route is the most prominent. Thus, a very small fraction of the substrate is converted to other hydroxylated and acidic metabolites. Although 2,2′-dihydroxybiphenyl is a poor substrate for strain B-356 biphenyl dioxygenase, metabolites were produced by the biphenyl catabolic enzymes, leading to production of 2-hydroxybenzoic acid. Data show that the major route of metabolism involves, as a first step, a direct dehydroxylation of one of the ortho-substituted carbons to yield 2,3,2′-trihydroxybiphenyl. However, other metabolites resulting from hydroxylation of carbons 5 and 6 of 2,2′-dihydroxybiphenyl were also produced, leading to dead-end metabolites.     

Metabolism of aromatic hydrocarbons by yeasts

Six yeasts were examined for their ability to metabolize naphthalene, biphenyl and benzo(a)pyrene. All of the organisms tested oxidized these aromatic hydrocarbons. Candida lipolytica oxidized naphthalene to 1-naphthol, 2-naphthol, 4-hydroxy-1-tetralone and trans-1,2-dihydroxy-1,2-dihydronaphthalene. The major metabolite was 1-naphthol. C. lipolytica oxidized biphenyl to produce 2-, 3-, and 4-hydroxybiphenyl, 4,4′-dihydroxybiphenyl and 3-methoxy-4-hydroxybiphenyl. 4-Hydroxybiphenyl was the predominant metabolite formed. C. lipolytica oxidized benzo(a)pyrene to 3-hydroxybenzo(a)pyrene and 9-hydroxybenzo(a)pyrene. Metabolites were isolated and identified by absorption spectrophotometry, mass spectrometry and thin-layer, gasliquid and high-pressure liquid chromatography. Where possible the structures of these metabolites were confirmed by comparison with authenic compounds.     

Metabolism of hydroxybiphenyl and chloro-hydroxybiphenyl by biphenyl/chlorobiphenyl degradingPseudomonas testosteroni,strain B-356

Summary A biphenyl (BP) and chlorobiphenyl (CBP) metabolizingPseudomonas testosteroni, strain B-356 was also capable of utilizing 2-, 3-, and 4-hydroxybiphenyl. Data presented here suggest that utilization of biphenyl and mono-subtituted biphenyls involves the enzymes of the same pathway. Chloro-hydroxybiphenyls were also metabolized by strain B-356. The unsubstituted ring is first hydroxylated in position 2 and 3 and then cleaved in ameta 1, and 2, position to ultimately generate the benzoic acid derivatives. Since strain B-356 was capable of utilizing benzoic acid and mono-hydroxybenzoic acids, the utilization of biphenyl, 2-, 3-, and 4-hydroxybiphenyl is complete at non-toxic concentrations of the substrates. Chlorobenzoic acids and chloro-hydroxybenzoic acids were not metabolized further by this strain. Studies usingPseudomonas putida, strain KT2440 carrying cloned BP/CBP genes from strain B-356 provided further evidence for the presence of a common pathway for the metabolism of the above compounds inP. testosteroni, strain B-356. Suggestions are made on significance of the broad substrate specificity of the enzymes of biphenyl/chlorobiphenyl pathway in regard to their possible origin and in relation to PCB mixture degradation.     

Transformation of ortho-substituted biphenyls by Methylosinus trichosporium OB3b: substituent effects on oxidation kinetics and product formation

The ability of Methylosinus trichosporium OB3b, expressing soluble methane monooxygenase, to oxidize a range of ortho-substituted biphenyls was examined to better understand how substituents affect both the rate and products of oxidation in comparison to biphenyl. Inhibition of oxidation was observed over the tested substrate range for both biphenyl and ortho-halogenated biphenyls (2-chloro-, 2-bromo-, and 2-iodobiphenyl). No inhibition was observed during the oxidation of 2-hydroxybiphenyl and 2-methylbiphenyl. Analysis of the products of oxidation showed that, depending on the substituent, ring hydroxylation, substituent oxidation, and elimination pathways could occur. The type and abundance of products formed along with the relatively high kinetic isotope effect observed for deuterated vs. nondeuterated biphenyl (k(h)/k(d) = 3.4+/-0.02) are consistent with mechanisms that include both hydrogen abstraction and NIH-shift pathways. Knowledge of these substituent-dependent reaction rates and mechanisms enhances our understanding of the methanotrophic aryl transformation potential and allows for better prediction of the formation of oxidized intermediates by methanotrophic bacteria.     

Metabolism of biphenyl by Aspergillus toxicarius: induction of hydroxylating activity and accumulation of water-soluble conjugates.

Biphenyl metabolism in Aspergillus toxicarius occurs by successive hydroxylations in the 4- and 4'-positions, followed by conjugation with sulfate to produce 4-hydroxybiphenyl-O-sulfonic acid and 4,4'-dihydroxybiphenyl-O-sulfonic acid. The hydroxylation reactions normally occur only after a prolonged lag period after which the appearance of the monohydroxylated compound precedes the dihydroxylated compound. The accumulation of the monohydroxy compound is transient; therefore, it is an intermediate in the hydroxylating pathway. The onset of hydroxylating activity can be greatly accelerated when the culture is primed with the intermediate or product of the reaction (4-hydroxybiphenyl or 4,4'-dihydroxybiphenyl) at the time of biphenyl addition; a concentration of 0.05 mg 4,4'-dihydroxybiphenyl per ml produces optimal induction. Water-soluble conjugates of 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl were found in cultures of A. toxicarius grown in the presence of biphenyl plus inducer. The conjugate was shown to be the sulfate ester; no glucuronide or other conjugate species was found in any phase of the transformation. As with hydroxylating activity, the sulfotransferase activity appeared to be induced by the products of biphenyl metabolism.     

Microbial Production of 4,4′-Dihydroxybiphenyl: Biphenyl Hydroxylation by Fungi

Of 15 species of fungi examined for their ability to hydroxylate biphenyl, 10 produced 4-hydroxybiphenyl. Seven of the 10 also produced 4,4′-dihydroxybiphenyl. The most efficient strains, Absidia pseudocylindrospora NRRL 2770 and Absidia sp. NRRL 1341, were more closely examined to determine their growth characteristics and the kinetics of biphenyl hydroxylation in batch fermentation. In the absence of biphenyl, A. pseudocylindrospora 2770 and Absidia sp. 1341 grew about as rapidly and efficiently in a defined glucose minimal medium as in a complex medium. Substrate yield coefficients for glucose in both media were 0.4 to 0.5 g of biomass per g of glucose, and the specific growth rate was about 0.17 h⁻¹ (doubling time, about 4 h). In this unoptimized system, 10 to 15 g of biomass per liter (dry weight) could be produced, using a defined salt solution and glucose as sole carbon and energy source. In the presence of biphenyl, growth was inhibited, more so for strain 1341 than for strain 2770. However, the specific activity for biphenyl hydroxylation (milligrams of biphenol per gram of biomass) was about 3.5 times greater for strain 1341. Furthermore, biphenyl hydroxylation appeared to require the presence of an oxidizable carbon and energy source (and perhaps growth) to proceed and, at least for strain 1341, hydroxylation seemed to be more efficient in the complex medium.     

Degradation of biphenyl by Mycobacterium sp. strain PYR-1

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.     

The metabolic pathway catalyzed by the tyrosinase of Agaricus bisporus

N-t-Butyloxycarbonyl-gamma-L-glutaminyl-2-bromo-4-hydroxybenzene alpha-benzyl ester was synthesized as a precursor to gamma-L-glutaminyl-4-hydroxy[2-3H]benzene. With this labeled compound and the previously synthesized gamma-L-glutaminyl-4-hydroxy[3,5-3H]benzene, the stoichiometry of ring substitution was determined for the tyrosinase-catalyzed metabolic pathway of Agaricus bisporus. In this pathway, gamma-L-glutaminyl-4-hydroxybenzene is hydroxylated to gamma-L-glutaminyl-3,4-dihydroxybenzene which is oxidized to gamma-L-glutaminyl-3,4-benzoquinone and a compound of previously unknown structure, "490." The results indicated that the "490" quinone was derived from gamma-L-glutaminyl-3,4-benzoquinone without further ring substitution. A base-catalyzed, nonenzymatic reaction of gamma-L-glutaminyl-3,4-benzoquinone was observed which yielded a compound with a 490 nm chromophore. gamma-Glutamyl transpeptidase cleavage of gamma-L-glutaminyl-3,4-dihydroxybenzene led to the release of 4-aminocatechol which air-oxidized to a compound with identical spectral properties to "490." The structure of "490" was thus determined to be 2-hydroxy-4-imino-2,5-cyclohexadiene-1-one(2-hydroxy-4-iminoquinone). The tyrosinase-catalyzed hydroxylation of gamma-L-glutaminyl-4-hydroxybenzene was found to be optimal at pH 8.0, while the enzymatic oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene was optimal at pH 6.0.