Elsevier

Gene

Volume 206, Issue 2, 12 January 1998, Pages 185-193
Gene

Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora

https://doi.org/10.1016/S0378-1119(97)00583-0Get rights and content

Abstract

A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I–P–X–P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5 kb contained the complete sequence of the Cs-mnp1 gene, including 162 bp and 770 bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT–AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9 kb was amplified using inverse PCR. A putative TATAA element was identified 5′ of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.

Introduction

Ceriporiopsis subvermispora is a white rot basidiomycete that efficiently degrades wood (Otjen et al., 1987) as well as grass lignocelluloses (Akin et al., 1995). Its ligninolytic system is composed of manganese peroxidase (MnP) and laccase (Rüttimann et al., 1992). The former is a heme-containing enzyme that oxidizes Mn(II) to Mn(III) (Glenn et al., 1986), which in turn oxidizes phenolic substrates in vitro and presumably lignin itself in natural environments. Laccase is a copper-containing phenoloxidase, although in the presence of appropriate substrates, it is able to attack non-phenolic compounds indirectly (Bourbannais and Paice, 1992). C. subvermispora does not produce lignin peroxidase (LiP), another heme protein possessing the unique ability to attack non-phenolic residues generating cation radicals that eventually decay to smaller compounds (Kirk et al., 1986). Recent reports indicate that in cultures of this fungus, MnP itself mediates the oxidation of non-phenolic lignin structures through a novel mechanism involving peroxidation of unsaturated lipids (Jensen et al., 1996).

MnP seems to be the most ubiquitous ligninolytic enzyme among white-rot fungi (Orth et al., 1993; Hatakka, 1994). In cultures of C. subvermispora, the titers of MnP correlate positively with mineralization of synthetic lignin (Rüttimann-Johnson et al., 1993). Analysis by isoelectrofocusing of samples withdrawn from the cultures reveals the presence of up to 11 MnPs, with an isoenzyme pattern that varies according to the conditions of growth (Lobos et al., 1994). The isoenzymes differ with respect to their substrate specificity and the concentration of Mn(II) required for optimal activity (Urzúa et al., 1995). N-terminal sequences of several of these MnP isoenzymes are clearly distinct, suggesting the existence of more than one gene (Lobos et al., 1994). The production of MnP as an enzyme family is not unique to C. subvermispora (Leisola et al., 1987; Johansson and Nyman, 1993; Rüttimann-Johnson et al., 1994; Périé et al., 1996), although the physiological significance of this multiplicity is not well understood.

Genetic studies constitute a suitable approach to gain insight into the latter phenomenon. Three genes for MnP have been identified in the fungus Phanerochaete chrysosporium (Godfrey et al., 1990; Mayfield et al., 1994; Alic et al., 1997), which are regulated in response to nutrient limitation, heat shock, concentration of Mn(II) and hydrogen peroxide (Pribnow et al., 1989; Brown et al., 1991, Brown et al., 1993; Mayfield et al., 1994; Li et al., 1995). In Trametes versicolor, N-terminal sequencing suggests the presence of three genes (Johansson et al., 1993), one of which has been recently sequenced at the nucleotide level (Johansson and Nyman, 1996). Two MnP cDNAs from the fungus IZU-154 (Matsubara et al., 1996) and a mnp gene from Pleurotus ostreatus (Asada et al., 1995) have also been sequenced.

The aim of our studies is to verify whether there is a gene family coding for the MnPs in C. subvermispora and to determine the conditions governing expression of selective isoenzymes. For this purpose, we have constructed both cDNA and genomic libraries of the fungus. This work reports the independent isolation and sequencing of a cDNA from one of the MnP isoenzymes and a Cs-mnp gene encoding the same protein.

Section snippets

Construction of a cDNA library in Lambda ZIPLOX

Nitrogen-frozen mycelium of C. subvermispora (strain FP105757, Forest Products Laboratory, Madison, WI) was blended under dry ice, and the frozen powder was added to 12.5 ml of homogenization solution (0.1% DEPC, 0.2 M Tris–HCl pH 8.5, 50 mM EGTA, 0.25 M NaCl, 0.6% p-aminosalysilic acid and 1% triisopropyl-naphthalenesulfonic acid) and 12.5 ml of water-saturated phenol. One gram of sterile glass beads (106 mm) (Glass Beads, Sigma) was added, and the mixture was incubated on ice for 60 min with

Cloning and sequencing of cDNA MnP13-1

A cDNA library was constructed in Lambda ZIPLOX using RNA isolated from C. subvermispora grown for 10 days in liquid cultures of defined composition (Rüttimann et al., 1992). The cDNA MnP13-1 clone was isolated by probing the library with a cDNA corresponding to MnP isoenzyme H3 from P. chrysosporium (Orth et al., 1994) as a probe. Subcloning and sequencing of the fragments yielded the complete sequence of this cDNA (Genbank accession no. U60413). Its characteristics are implicit in Fig. 1,

Acknowledgements

This work was financed by grant 1971239 from FONDECYT, Chile.

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