1,4-Benzoquinone Reductase from Phanerochaete chrysosporium: cDNA Cloning and Regulation of Expression

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Applied and Environmental Microbiology


Biological Sciences







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A cDNA clone encoding a quinone reductase (QR) from the white rot basidiomycete Phanerochaete chrysosporium was isolated and sequenced. The cDNA consisted of 1,007 nucleotides and a poly(A) tail and encoded a deduced protein containing 271 amino acids. The experimentally determined eight-amino-acid N-terminal sequence of the purified QR protein from P. chrysosporium matched amino acids 72 to 79 of the predicted translation product of the cDNA. The M r of the predicted translation product, beginning with Pro-72, was essentially identical to the experimentally determined M r of one monomer of the QR dimer, and this finding suggested that QR is synthesized as a proenzyme. The results of in vitro transcription-translation experiments suggested that QR is synthesized as a proenzyme with a 71-amino-acid leader sequence. This leader sequence contains two potential KEX2 cleavage sites and numerous potential cleavage sites for dipeptidyl aminopeptidase. The QR activity in cultures of P. chrysosporium increased following the addition of 2-dimethoxybenzoquinone, vanillic acid, or several other aromatic compounds. An immunoblot analysis indicated that induction resulted in an increase in the amount of QR protein, and a Northern blot analysis indicated that this regulation occurs at the level of theqr mRNA.

The wood-rotting basidiomycete fungus Phanerochaete chrysosporium degrades polymeric lignin (12, 21, 30) and various aromatic pollutants, including chlorinated phenols, dioxins, and polyaromatic hydrocarbons (9, 23, 26, 40). A wide variety of oxidized metabolic intermediates are generated during the degradation of lignin, lignin model compounds, and aromatic pollutants by this organism. These intermediates include substituted quinones, hydroquinones, benzaldehydes, benzoic acids, and ring-opened fragments (12, 15, 27, 30), which are metabolized further by intracellular processes (7, 42, 55). The extracellular peroxidases that are involved in the initial oxidative steps of lignin and pollutant degradation are well-characterized (15, 21, 27, 30); however, much less is known about the intracellular enzymes involved in the further degradation of monomeric intermediates, such as quinones, hydroquinones, and benzaldehydes. Recent work, including the elucidation of metabolic pathways for the degradation of several aromatic pollutants by P. chrysosporium (53,54), has suggested that intracellular enzymes are involved in the reduction of quinones. Since benzoquinones are generated by the peroxidase-catalyzed oxidation of lignin and appear to be key intermediates in the degradation of aromatic compounds by P. chrysosporium (29, 42, 44, 51, 53, 54), we have been examining the reduction of benzoquinones by this organism.

The reduction of methoxylated, lignin-derived quinones by P. chrysosporium appears to be catalyzed by an intracellular quinone reductase (QR) or QRs (6, 7, 11, 13), and we have purified and partially characterized one intracellular, NAD(P)H-dependent QR from this organism (6, 7). The soluble protein is a 44-kDa dimer with two similar 22-kDa subunits and appears to contain two flavin mononucleotide (FMN) moieties per dimer. A variety of methoxylated quinones and other electron acceptors serve as substrates for this enzyme (6, 7). The stoichiometry of NADH oxidation to 2,6-dimethoxy-1,4-benzoquinone (DMBQ) reduction is 1:1, and the enzyme apparently catalyzes the reduction of quinones to hydroquinones via a ping-pong, steady-state, kinetic mechanism (6). NADH and NADPH are equally efficient as electron donors, and the enzyme is competitively inhibited, with respect to NADH, by both dicoumarol and Cibacron Blue (6). The latter properties are similar to properties reported for the mammalian QR DT-diaphorase (16,17, 37).

Here, we describe the isolation and characterization of a cDNA clone encoding the P. chrysosporium QR, as well as studies on the regulation of QR synthesis.