Study of Biochemical Pathways and Enzymes Involved in Pyrene Degradation by Mycobcterium sp. Strain KMS
Applied and Environmental Microbiology
Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, and 4-phenanthroic acid, were identified during pyrene degradation. Pyrene-4,5-dione, which accumulates as an end product in some gram-negative bacterial cultures, was further utilized and degraded by Mycobacterium sp. strain KMS. Enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS were studied, using 2-D gel electrophoresis. The first protein in the catabolic pathway, aromatic-ring-hydroxylating dioxygenase, which oxidizes pyrene to cis-4,5-pyrene-dihydrodiol, was induced with the addition of pyrene and pyrene-4,5-dione to the cultures. The subcomponents of dioxygenase, including the alpha and beta subunits, 4Fe-4S ferredoxin, and the Rieske (2Fe-2S) region, were all induced. Other proteins responsible for further pyrene degradation, such as dihydrodiol dehydrogenase, oxidoreductase, and epoxide hydrolase, were also found to be significantly induced by the presence of pyrene and pyrene-4,5-dione. Several nonpathway-related proteins, including sterol-binding protein and cytochrome P450, were induced. A pyrene degradation pathway for Mycobacterium sp. strain KMS was proposed and confirmed by proteomic study by identifying almost all the enzymes required during the initial steps of pyrene degradation.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants. Bioremediation is broadly accepted as an effective tool for the degradation of PAHs to nontoxic compounds. Pyrene degradation pathways of mycobacteria, including Mycobacterium vanbaalenii PYR-1, M. flavescens PYR-GCK, Mycobacterium sp. strain RJGII-135, Mycobacterium sp. strain KR2, and Mycobacterium sp. strain AP1, have been studied and are proposed to be similar (3, 4, 16, 26, 28, 29). The proposed pathway is thought to be catalyzed by a number of enzymes. Pyrene is first oxidized in the K region by a dioxygenase to form cis-4,5-pyrene-dihydrodiol, which is rearomatized to form 4,5-dihydroxy-pyrene by dihydrodiol dehydrogenase. 4,5-Dihydroxy-pyrene is subsequently cleaved to yield phenanthrene-4,5-dicarboxylic acid by intradiol dioxygenase. Following loss of a carboxyl group by decarboxylase, 4-phenanthroic acid is formed. Oxidation of 4-phenanthoic acid by ring-hydroxylating dioxygenase produces 3,4-phenanthrene dihydrodiol-4-carboxylic acid, which is further transformed to 3,4-dihydroxyphenanthrene by dehydrogenase/decarboxylase. Once 3,4-dihydroxyphenanthrene is formed, it enters the phenanthrene degradation pathway (18). Two additional pathways have been proposed. One proposition is that pyrene hydroxylation takes place at the 1, 2 positions, leading to the formation of 4-hydroxy-perinaphthenone, which is a dead end product and so far has been found only in M. vanbaalenii PYR-1 cultures (3). Another pathway involves the accumulation of 6,6′-dihydroxy-2,2′-biphenyl-dicarboxylic acid in Mycobacterium sp. strain AP1 (29).
Pyrene-4,5-dione can be formed following the autooxidation of 4,5-dihydroxy-pyrene and is a pyrene degradation metabolite in several bacteria. It was observed as a pyrene metabolite accumulated in Sphingomonas yanoikuyae strain R1 (12). Significant amounts of this compound were formed when M. vanbaalenii PYR-1 was incubated with a high concentration of cis-4,5-pyrene-dihydrodiol, although it was not reported as an intermediate when M. vanbaalenii PYR-1 grew on pyrene (12). In addition, pyrene-4,5-dione accumulation was observed in aged whole-sediment microcosm incubations (6) and in slurry-phase reactors with soil suspension at 25% wt/vol (5). Moreover, it was identified to be a pyrene metabolite in the phagemid clone My6-pBK-CMV, which contained a dioxygenase gene when it was incubated with pyrene (13). Even though pyrene 4,5-dione is a growth substrate for M. vanbaalenii PYR-1 (12), its further degradation is not delineated.
Based on the proposed metabolic pathway for the degradation of pyrene and phenanthrene by Mycobacterium species (18), at least 15 enzymes are involved in the degradation of pyrene and the o-phthalate degradation of phenanthrene, with some enzymes being common to the degradation of both PAHs. While some of the enzymes, including dioxygenase, aldehyde dehydrogenase, putative monooxgenase, hydratase aldolase, and catalase-peroxidase (14, 18, 30), have been identified as PAH-induced proteins, some other enzymes, especially those involved in the initial steps of pyrene degradation, are not known.
Mycobacterium sp. strain KMS was isolated from vadose-zone soil of the Champion International Superfund site (Libby, MT) and has the ability to degrade pyrene and other PAHs (22). The genome was sequenced by the U.S. Department of Energy/Joint Genome Institute (JGI), and the draft sequence is available in the NCBI database. Due to the toxicity of pyrene-4,5-dione (25) and its possible presence during pyrene degradation in mycobacteria, it is important to identify this metabolite and determine its fate during pyrene degradation, as it was observed as an end product in some gram-negative cultures and may result in an increase in toxicity during in situ soil bioremediation (12). Furthermore, biostimulation and bioaugmentation strategies, where Mycobacterium sp. strain KMS is employed for PAH bioremediation, require an understanding of enzymatic mechanisms. Therefore, the objectives of this work reported here were (i) to determine the pyrene degradation pathway used by Mycobacterium sp. strain KMS by isolating and identifying the metabolites, (ii) to determine the capability of Mycobacterium sp. strain KMS to degrade pyrene-4,5-dione, (iii) to obtain the proteomic profile of Mycobacterium sp. strain KMS, and (iv) to identify PAH-induced proteins.
Liang, Y.; Gardner, D.; Miller, C.; Chen, Dong; Anderson, A.; Weimer, B.; and Sims, Ronald C., "Study of Biochemical Pathways and Enzymes Involved in Pyrene Degradation by Mycobcterium sp. Strain KMS" (2006). Biological Engineering Faculty Publications. Paper 10.