Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius.
Aryl-alcohol oxidase (AAO) involved in lignin degradation by Pleurotus pulmonarius has been purified and characterized. The enzyme was produced in glucose-peptone medium and isolated in a sole chromatographic step using Sephacryl 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-anisyl alcohol, with constants similar to those of Pleurotus eryngii and Bjerkandera adusta AAO but different from the intracellular AAO described in Phanerochaete chrysosporium, which present the highest activity on m-anisyl alcohol. Simultaneously, the cDNA of P. pulmonarius AAO has been cloned and sequenced. The translation of this sequence consisted of 593 amino acids including a signal peptide of 27 amino acids. The comparison with other alcohol oxidases, 35% amino acid identity with glucose oxidase, showed highly conserved amino acid sequences in N-terminal and C-terminal regions, in spite of differences in substrate specificity. Crystallization of AAO, carried out for the first time using the P. pulmonarius enzyme, will permit to obtain a molecular model for this oxidase and establish some characteristic of its catalytic site and general structure.
Varela
E
E
Centro de Investigaciones Biológicas, CSIC, Velázquez 144, E-28006, Madrid, Spain.
Böckle
B
B
Romero
A
A
Martínez
A T
AT
Martínez
M J
MJ
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018076:DNA, Complementary / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D018076:DNA, Complementary; D005656:Fungal Proteins; D000429:Alcohol Oxidoreductases; C077759:aryl-alcohol oxidase