A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix and associates with p68 RNA helicase.

The cell nucleus is structurally and functionally organized by the nuclear matrix. We have examined whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for targeting to the subnuclear compartment and for interaction with other proteins. AKAP95 was expressed in mammalian cells and found to localize exclusively to the nuclear matrix. Mutational analysis was used to identify determinants for nuclear localization and nuclear matrix targeting of AKAP95. These sites were found to be distinct from previously identified DNA and protein kinase A binding domains. The nuclear matrix-targeting site is unique but conserved among members of the AKAP95 family. Direct binding of AKAP95 to isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting site. Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix isolated from rat brain. Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase. The helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were precipitated as a complex from solubilized cell extracts. The results define novel protein-protein interactions among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly of hormonally responsive transcription complexes.

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

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.

Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium.

The gene encoding manganese peroxidase isozyme 3 (MnP3) from the white-rot basidiomycete Phanerochaete chrysosporium was cloned and sequenced. The mnp3 gene encodes a mature protein of 357 amino acids with a 25 amino-acid signal peptide. The amino acids involved in peroxidase function, as well as those forming the MnII binding site and those involved in disulfide bond formation, are conserved in the MnP3 sequence. The mnp3 gene has six introns, indicating that the sequenced P. chrysosporium mnp genes can be divided into three subfamilies on the basis of intron-exon structure. The mnp3 gene promoter contains putative metal response elements and heat shock elements which may be involved in the regulation of mnp gene transcription by Mn, the substrate for the enzyme, and by heat shock.

A reporter gene construct for studying the regulation of manganese peroxidase gene expression.

The orotidylate decarboxylase (ODase) gene (ura1) from Schizophyllum commune was utilized as a reporter for studying Mn regulation of the manganese peroxidase (MnP) gene (mnp) from the lignin-degrading basidiomycete Phanerochaete chrysosporium. A 1,500-bp fragment of the mnp1 promoter was fused upstream of the coding region of the ODase gene in a plasmid (pAMO) containing the S. commune ade5 gene as a selectable marker. pAMO was used to transform a P. chrysosporium ade1 ura11 mutant lacking endogenous ODase activity. When the P. chrysosporium transformant was grown in nitrogen-limited, Mn(II)-sufficient cultures, ODase activity was detected only during secondary metabolic growth and the pattern of ODase expression was similar to that of endogenous MnP. When Mn was added to 6-day-old nitrogen-limited, Mn-deficient cultures, both ODase activity and MnP activity were induced synchronously with maximal activity at 30 h. Growth in high-nitrogen-concentration medium suppressed the induction of both the ODase and endogenous MnP. These results indicate that this promoter-reporter construct can be used to study the regulation of the mnp gene.

Gene replacement in the lignin-degrading basidiomycete Phanerochaete chrysosporium.

The ability to carry out gene replacements and gene targeting in the lignin-degrading basidiomycete fungus, Phanerochaete chrysosporium, would facilitate studies on the roles and regulation of various components of its lignindegrading system. A plasmid consisting of the P. chrysosporium ura3 gene (encoding orotidylate decarboxylase) interrupted with the Schizophyllum commune ade2 gene (encoding an adenine biosynthetic enzyme) was used to transform the P. chrysosporium ade2 strain to adenine prototrophy with selection on 5-fluoroorotic acid for inactivation of the ura3 gene. Stable Ade+Ura- strains were obtained at a frequency of approximately one transformant per microgram of DNA. In all of the Ade+Ura- transformants examined by Southern analysis, the chromosomal ura3 locus had been replaced by the plasmid insert.

Isolation and transformation of uracil auxotrophs of the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Uracil auxotrophs of Phanerochaete chrysosporium were isolated using 5-fluoroorotate resistance as a selection scheme. The ura3 auxotrophs deficient in orotidylate decarboxylase and ura5 auxotrophs deficient in orotate phosphoribosyl transferase were characterized by enzyme assays and complementation tests. The ura5 auxotrophs were transformed to prototrophy with the ura5 gene from the ascomycete Podospora anserina. The ura3 auxotrophs were transformed to prototrophy with the ura3 gene from the basidiomycete Schizophyllum commune. The P. chrysosporium ura3 gene was isolated from a lambda EMBL3 genomic library using the S. commune ura3 gene as a probe. A 6.6-kb fragment incorporating the ura3 gene was subcloned into Bluescript SK+(pURA3.1) and used to transform P. chrysosporium ura3 auxotrophic strains. The pURA3.1 insert was mapped for restriction sites and the approximate location of the ura3 gene within the insert was determined. Double auxotrophic strains were transformed with either of two marker genes and the resulting single auxotrophic strains were crossed to demonstrate genetic recombination between two nuclei of identical genetic background.

Lignin peroxidase from the basidiomycete Phanerochaete chrysosporium is synthesized as a preproenzyme.

The cDNA clone L18 encoding lignin peroxidase LiP2, the most highly expressed LiP isozyme from Phanerochaete chrysosporium strain OGC101, was isolated and sequenced. Comparison of the cDNA sequence with the N-terminal sequence of the mature LiP2 protein isolated from culture medium suggests that the mature protein contains 343 amino acids (aa) and is preceded by a 28-aa leader sequence. In vitro transcription followed by in vitro translation and processing by signal peptidase resulted in cleavage at a site following the Ala21 (counted from the N-terminal Met1 of the initial translation product). The resultant protein contains a 7-aa propeptide, indicating that LiP is synthesized as a preproenzyme.

Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium: spectral characterization of the oxidized states and the catalytic cycle.

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the Mn(II)-dependent oxidation of a variety of phenols. Herein, we spectroscopically characterize the oxidized states of MnP compounds I, II, and III and clarify the role of Mn in the catalytic cycle of the enzyme. Addition of 1 equiv of H2O2 to the native ferric enzyme yields compound I, characterized by absorption maxima at 407, 558, 605, and 650 nm. Addition of 2 or 250 equiv of H2O2 to the native enzyme yields compound II or III, respectively, identified by absorption maxima at 420, 528, and 555 nm or at 417, 545, and 579 nm, respectively. These characteristics are very similar to those of horseradish peroxidase (HRP) and lignin peroxidase (LiP) compounds I, II, and III. Addition of 1 equiv of either Mn(II), ferrocyanide, or a variety of phenols to MnP compound I rapidly reduces it to MnP compound II. In contrast, only Mn(II) or ferrocyanide, added at a concentration of 1 equiv, reduces compound II. The Mn(III) produced by the enzymic oxidation of Mn(II) oxidizes the terminal phenolic substrates. This indicates that compounds I and II of MnP contain 2 and 1 oxidizing equiv, respectively, over the native ferric resting enzyme and that the catalytic cycle of the enzyme follows the path native enzyme----compound I----compound II----native enzyme. In addition, these results indicate that Mn(II) serves as an obligatory substrate for MnP compound II, allowing the enzyme to complete its catalytic cycle. Finally, the Mn(II)/Mn(III) redox couple enables the enzyme to rapidly oxidize the terminal phenolic substrates.

Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium.

The manganese peroxidase (MnP), from the lignin-degrading fungus Phanerochaete chrysosporium, an H2O2-dependent heme enzyme, oxidizes a variety of organic compounds but only in the presence of Mn(II). The homogeneous enzyme rapidly oxidizes Mn(II) to Mn(III) with a pH optimum of 5.0; the latter was detected by the characteristic spectrum of its lactate complex. In the presence of H2O2 the enzyme oxidizes Mn(II) significantly faster than it oxidizes all other substrates. Addition of 1 M equivalent of H2O2 to the native enzyme in 20 mM Na-succinate, pH 4.5, yields MnP compound II, characterized by a Soret maximum at 416 nm. Subsequent addition of 1 M equivalent of Mn(II) to the compound II form of the enzyme results in its rapid reduction to the native Fe3+ species. Mn(III)-lactate oxidizes all of the compounds which are oxidized by the enzymatic system. The relative rates of oxidation of various substrates by the enzymatic and chemical systems are similar. In addition, when separated from the polymeric dye Poly B by a semipermeable membrane, the enzyme in the presence of Mn(II)-lactate and H2O2 oxidizes the substrate. All of these results indicate that the enzyme oxidizes Mn(II) to Mn(III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds. In the absence of exogenous H2O2, the Mn-peroxidase oxidized NADH to NAD+, generating H2O2 in the process. The H2O2 generated by the oxidation of NADH could be utilized by the enzyme to oxidize a variety of other substrates.