1,4-benzoquinone reductase from Phanerochaete chrysosporium: cDNA cloning and regulation of expression

Abstract

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 Mr of the predicted translation product, beginning with Pro-72, was essentially identical to the experimentally determined Mr 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 the qr mRNA.

FIG. 1

Nucleotide sequence of the P. chrysosporium qr cDNA. The amino acid sequence of the predicted translation product is shown below the nucleotide sequence. The qr coding region is flanked by a 59-bp 5′ noncoding region and a 132-bp 3′ noncoding region, excluding the poly(A) tail. The initiation codon is followed by an apparent 71-amino-acid leader sequence, which is underlined. The experimentally determined N-terminal amino acid sequence of the isolated QR protein is overlined. The two KEX2 cleavage sites in the leader sequence are enclosed in boxes. The dots indicate the potential glycosylation sites at residues 11 and 223.

FIG. 2

SDS-PAGE of the in vitro transcription-translation products obtained with the qr cDNA. Reactions and electrophoresis were performed as described in the text. Lane 1, control reaction mixture lacking DNA; lane 2, reaction mixture containing pQRT1, which included the upstream ATG of pcQR1; lane 3, reaction mixture containing pQRT2, which included only the downstream ATG of pcQR1. The prestained molecular mass markers used were carbonic anhydrase (34.8 kDa), soybean trypsin inhibitor (28.3 kDa), and lysozyme (20.4 kDa).

FIG. 3

Time course for induction of QR activity. Vanillic acid (■), MBQ (▴), or nothing (•) was added to 2-day-old HN cultures, as described in the text. Induced and uninduced cells were harvested at the times indicated, and the QR activity in the supernatants of crude extracts was assayed as described in the text.

FIG. 4

Induction of QR activity with different concentrations of vanillic acid. Two-day-old HN cultures were induced with the indicated concentrations of vanillic acid. The cells were harvested after 16 h and broken, and QR activity was measured as described in the text.

FIG. 5

Immunoblot analysis of QR from uninduced and vanillic acid-induced cells. Two-day-old HN cultures were not induced (lane 1) or were induced with 0.5 mM (lane 2) or 2.0 mM (lane 3) vanillic acid and then were harvested after 16 h, as described in the text. Portions (50 μg) of the supernatant proteins from crude cell extracts were loaded and electrophoresed on SDS-PAGE gels, transferred to nitrocellulose, and immunodetected, as described in the text.

FIG. 6

Northern blot of P. chrysosporium RNA probed with the qr cDNA and the gpd gene. Two-day-old HN cultures were not induced (lane 1) or were induced with vanillic acid (2 mM) (lane 2) or MBQ (0.2 mM) (lane 3), as described in the text. After 3 h, RNA were isolated from induced and uninduced cultures, electrophoresed, and transferred to membranes, as described in the text. The blots were probed with randomly primed ³²P-labeled pcQR1, as described in the text. Then the blots were stripped and reprobed with ³²P-labeled gpd.

FIG. 7

Clustal alignment of deduced protein sequences derived from the QR gene (this work), the brefeldin A resistance gene (bre) from S. pombe (52), and a gene (alt) encoding a minor allergen from Alternaria alternata (1). Identical portions of the sequences (bold) are enclosed in boxes; shading indicates sequence similarity.