Downloaded from www.microbiologyresearch.org by IP: 54.147.156.167 On: Thu, 02 Jun 2016 15:26:54 Oxalate decarboxylase of the white-rot fungus Dichomitus squalens demonstrates a novel enzyme primary structure and non-induced expression on wood and in liquid cultures Miia R. Ma ¨ kela ¨ , Kristiina Hilde ´ n, Annele Hatakka and Taina K. Lundell Correspondence Miia R. Ma ¨ kela ¨ miia.r.makela@helsinki.fi Department of Applied Chemistry and Microbiology, Division of Microbiology, Viikki Biocenter, PO Box 56, FIN-00014 University of Helsinki, Finland Received 3 March 2009 Revised 20 April 2009 Accepted 21 April 2009 Oxalate decarboxylase (ODC) catalyses the conversion of oxalic acid to formic acid and CO 2 in bacteria and fungi. In wood-decaying fungi the enzyme has been linked to the regulation of intra- and extracellular quantities of oxalic acid, which is one of the key components in biological decomposition of wood. ODC enzymes are biotechnologically interesting for their potential in diagnostics, agriculture and environmental applications, e.g. removal of oxalic acid from industrial wastewaters. We identified a novel ODC in mycelial extracts of two wild-type isolates of Dichomitus squalens, and cloned the corresponding Ds-odc gene. The primary structure of the Ds-ODC protein contains two conserved Mn-binding cupin motifs, but at the N-terminus, a unique, approximately 60 aa alanine-serine-rich region is found. Real-time quantitative RT-PCR analysis confirmed gene expression when the fungus was cultivated on wood and in liquid medium. However, addition of oxalic acid in liquid cultures caused no increase in transcript amounts, thereby indicating a constitutive rather than inducible expression of Ds-odc. The detected stimulation of ODC activity by oxalic acid is more likely due to enzyme activation than to transcriptional upregulation of the Ds-odc gene. Our results support involvement of ODC in primary rather than secondary metabolism in fungi. INTRODUCTION Oxalic acid is the predominant organic acid produced by wood-rotting fungi when they are cultivated on defined liquid media or on solid lignocelluloses (Kuan & Tien, 1993; Shimada et al., 1997; Galkin et al., 1998; Urzu ´a et al., 1998; Hofrichter et al., 1999; Ma ¨kela ¨ et al., 2002). According to the type of decay that they cause on wood, these organisms may be classified as white-, brown- and soft-rot fungi (Kuan & Tien, 1993; Hatakka, 2001). Fungi synthesize oxalic acid in their mitochondria as a waste compound from the tricarboxylic acid cycle, and by the glyoxylate cycle that operates in the glyoxysomes (Espejo & Agosin, 1991; Dutton & Evans, 1996; Munir et al., 2001). Typically, brown-rot fungi produce high quantities of extracellular oxalic acid (Dutton et al., 1993; Espejo & Agosin, 1991), although it has been shown that both white- and brown-rot fungi express specific oxalate- degrading enzymes (Mehta & Datta, 1991; Dutton et al., 1994; Micales, 1997; Aguilar et al., 1999; Ma ¨kela ¨ et al., 2002). Three types of oxalate-degrading enzymes have been described in microbes and plants: oxalate decarboxylases (ODC, EC 4.1.1.2), oxalate oxidases (OXO, EC 1.2.3.4) and oxalyl-CoA decarboxylases (OXC, EC 4.1.1.8) (Svedruz ˇic ´ et al., 2005). ODC, isolated from fungi and bacteria, is a Mn-containing enzyme that decomposes oxalic acid to formic acid and CO 2 in a reaction that requires O 2 (Reinhardt et al., 2003). ODCs belong to the cupin protein superfamily, characterized by a conserved metal-ion- binding cupin motif with an overall b-barrel fold (Dunwell et al., 2000, 2004), and are further classified as bicupins, as ODCs possess two cupin motifs, probably due to an evolutionary gene duplication event (Dunwell et al., 2004). The evolutionarily related monocupin enzyme OXO similarly requires O 2 but cleaves oxalic acid to two CO 2 with generation of H 2 O 2 . OXO is expressed mainly in plants; only one fungal OXO has been reported so far (Aguilar et al., 1999). The third enzyme, OXC, is a bacterial enzyme, which converts activated oxalyl-CoA to formyl- Abbreviations: FDH, formate dehydrogenase; FPLC, fast protein liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ODC, oxalate decarboxylase; OXC, oxalyl-CoA decarboxylase; OXO, oxalate oxidase; qRT-PCR, quantitative RT-PCR; UTR, untranscribed region. The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are FM946037, FM955140, FM946036, and FM954981 for odc, cDNA of odc, partial gapdh of D. squalens FBCC312, and odc of D. squalens FBCC184, respectively. Microbiology (2009), 155, 2726–2738 DOI 10.1099/mic.0.028860-0 2726 028860 G 2009 SGM Printed in Great Britain