BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Structural characterization of heterodimeric laccases from Pleurotus ostreatus Paola Giardina & Flavia Autore & Vincenza Faraco & Giovanna Festa & Gianna Palmieri & Alessandra Piscitelli & Giovanni Sannia Received: 21 February 2007 / Revised: 15 March 2007 / Accepted: 16 March 2007 / Published online: 12 April 2007 # Springer-Verlag 2007 Abstract The subfamily of POXA3 laccase isoenzymes produced by the fungus Pleurotus ostreatus has been characterized as an example of the complexity and hetero- geneity of fungal isoenzyme patterns. Two isoenzymes, POXA3a and POXA3b, were previously purified, exhibiting an unusual heterodimeric structure composed of a large (67 kDa) and a small (18 or 16 kDa) subunit. A unique gene encodes the large subunit of both POXA3a and POXA3b, but alternative splicing produces two variants—differing for an insertion of four amino acids—for each isoenzyme. Two genes encoding POXA3a and POXA3b small subunits have been identified, and the corresponding amino acid sequences show only two amino acid substitutions. The 18- and 16-kDa subunits of both POXA3a and POXA3b differ for N- glycosylation at Asn150 of the 16-kDa subunit. The POXA3 large subunit 3D model allows us to highlight peculiarities of this molecule with respect to the laccases whose 3D structures are known. Keywords Phenol oxidase . White rot fungi . Quaternary structure Introduction Laccases are multicopper oxidative enzymes, useful for biotransformation of environmental organic pollutants and exploited industrially in various oxidative processes. Mul- tiple isoforms of laccases are usually secreted by each fungus depending on species and environmental conditions. Up to 17 laccase-encoding genes have been found in the genome of Coprinopsis cinerea (Kilaru et al. 2006). Laccases are the major extracellular components of the lignin-degrading system of the white rot fungi belonging to the Pleurotus genus (Baldrian et al. 2005). The isoenzymes produced by Pleurotus ostreatus have been extensively studied. POXC laccase is the most abundantly produced under all the growth conditions examined (Palmieri et al. 1993); other isoenzymes secreted by the mycelium have also been purified and characterized (POXA1w and POXA1b) (Palmieri et al. 1997; Giardina et al. 1999). Two closely related laccase isoenzymes (POXA3a and POXA3b) produced by P. ostreatus in copper supplemented cultures were purified (Palmieri et al. 2003). They exhibit unusual structural features. Unlike most of the known laccases that are monomeric proteins, both native isoenzymes are heterodimers constituted of a large (67 kDa) and a small (18- or 16-kDa) subunit (Palmieri et al. 2003). Some laccases endowed with quaternary structure have already been found in Phellinus ribis (Min et al. 2001), Trametes villosa (Yaver et al. 1996), Pleurotus pulmonarius (De Souza and Peralta 2003), and Rhizoctonia solani (Wahleithner et al. 1996), but all of them are homodimeric proteins. A few heterooligo- meric laccases were found in the fungi Monocillium indicum (Thakker et al. 1992), Agaricus bisporus (Perry et al. 1993) and Armillaria mellea (Curir et al. 1997), but only limited characterization of these proteins was reported. Appl Microbiol Biotechnol (2007) 75:1293–1300 DOI 10.1007/s00253-007-0954-4 P. Giardina (*) : F. Autore : V. Faraco : G. Festa : A. Piscitelli : G. Sannia Dipartimento di Chimica Organica e Biochimica, Università di Napoli “Federico II,” Complesso Universitario Monte S. Angelo, via Cintia, Naples 80126, Italy e-mail: giardina@unina.it G. Palmieri IBP, Consiglio Nazionale delle Ricerche, via P. Castellino 111, Naples 80131, Italy