Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius

Aryl-alcohol oxidase (AAO) involved in lignin degradation by Pleurotus pulm onarius has been purified and characterized. The enzyme was produced in glu cose-peptone medium and isolated in a sole chromatographic step using Sepha cryl S-200. The purified enzyme is an extracellular glycoprotein with 14% N -carbohydrate content and an estimated molecular mass of 70.5 kDa and pi of 3.95. The kinetic studies showed the highest enzyme affinity against p-ani syl alcohol, with constants similar to those of Pleurotus eryngii and Bjerk andera adusta AAO but different from the intracellular AAO described in Pha nerochaete chrysosporium, which present the highest activity on m-anisyl al cohol. Simultaneously, the cDNA of P. pulmonarius AAO has been cloned and s equenced. The translation of this sequence consisted of 593 amino acids inc luding a signal peptide of 27 amino acids. The comparison with other alcoho l oxidases, 35% amino acid identity with glucose oxidase, showed highly con served amino acid sequences in N-terminal and C-terminal regions, in spite of differences in substrate specificity. Crystallization of AAO, carried ou t for the first time using the P. pulmonarius enzyme, will permit to obtain a molecular model for this oxidase and establish some characteristic of it s catalytic site and general structure. (C) 2000 Elsevier Science B.V. All rights reserved.