Aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Gene cloning, sequence analysis, expression, and purification of the recombinant enzyme.
A cDNA clone encoding a ligninolytic aryl-alcohol dehydrogenase (AAD; EC 1.1.1.91) from the white-rot basidiomycete fungus Phanerochaete chrysosporium was isolated and characterized. The nucleotide sequence obtained reveals an open reading frame encoding a protein of 385 amino acids. Substantial homology (49.3% identity and 67.3% similarity, respectively) was observed between AAD and an open reading frame sequence present on chromosome III of Saccharomyces cerevisiae. A Southern blot analysis showed the presence of multiple AAD gene-related sequences in P. chrysosporium and in other white-rot fungi including Bjerkandera adusta and Fomes lignosus. Northern blot analyses are in line with the view that the levels and appearance of AAD mRNA correlate with the level and appearance of AAD activity and that, under conditions of nitrogen limitation, the AAD mRNA levels are higher than in carbon limited cultures. This is consistent with the regulation of the enzyme by carbon or nitrogen limitation being at the level of transcription. Moreover, the appearance of AAD-specific transcripts correlates with the appearance of lignin peroxidase-specific transcripts in the same cultures. This co-appearance is in line with the proposed synergistic interaction of the two enzymes in lignin biodegradation, which suggests a similar regulation. The AAD encoding cDNA was expressed in Escherichia coli to yield high levels of active enzyme, and the recombinant enzyme was purified by using metal chelate affinity chromatography.
Reiser
J
J
Institut für Biotechnologie, Eidgenössiche Technische Hochschule, ETH-Hönggerberg, Zürich, Switzerland.
Muheim
A
A
Hardegger
M
M
Frank
G
G
Fiechter
A
A
D000363:Agaricales / Q000201:enzymology* / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C055882:aryl-alcohol dehydrogenase