Aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Gene cloning, sequence analysis, expression, and purification of the recombinant enzyme.

J Reiser,A Muheim,M Hardegger,G Frank,A Fiechter

doi:10.1016/s0021-9258(18)46907-4

Abstract

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.

Highlights

From the Institut furBiotechnologie and lhstitut furMolekularbiologie und Biophysik, Eidgenossiche llkchnische Hochschule, ETH-Honggerberg, CH-8093 Zurich, Switzerland
A cDNA cloneencodingaligninolyticaryl-alcohol degradation, subsequent studies indicated the need for dehydrogenase ( A A D ; EC 1.1.1.91) fromthewhite-rot additional enzymes(reviewed by KirkandFarrell (1987), basidiomycetefungus Phanerochaete chrysosporium Schoemaker et al (19891, Pease and Tien (1991), Cullen and wasisolatedandcharacterizedT. henucleotide se- Kersten (1992), and Fiechter(1993))
The constantoxidation of quence obtained reveals an open reading frame encod-lignin by peroxidases will end up ian very oxidized state of the ing a proteinof 386 amino acids

Summary

THEJOURNALOF BIOLOGICAHLEMISTRY

Vol 269, No 45, Issue of November 11, pp. 28152-28159, 1994 Printed in U.S.A. GENE CLONING, SEQUENCE ANALYSIS, EXPRESSION, AND PURIFICATION OF THE RECOMBINANT ENZYME*. Reacted all positively with the dehydrogenase,but antibodies purified DNA Isolation and Southern Blot Analysis-Total DNA from P. chryon fusion proteins of clones obtained from library 1did not detect the sosporium, B. adusta, C. versicolor, P. ostreatus, and F: lignosus was dehydrogenase in Western blots.High titer phage lysates were pre- isolated essentially as described by Raeder and Broda (1988). One of them was initially prepared usingRNA isolated from 6-day-old carbon-limited cultures (library 1;Walther et al, 19881, while the other one had been prepared usinRgNA from 5- and 6-day-old nitrogen-limited cultures(library 2; Pribnow et al, 1989) Both libraries were screened through two cycles using polyclonal antibodies directed againsAt AD (Muheimet al., 1991).To compare the immunopositive phage clones at the molecular level, the phage DNAs were isolated, digested with EcoRI, and analyzed in a Southern blot. In order todemonstratea correlation between the protein encoded by the cDNA and the fungus-derived enzyme predicted AAD protein encoded by the cDNA and the fungus- correlate as far as this qualitative amino acid fingerprinting derived enzyme, the blot with the 43-kDa AAD from l? chry- method is concerned

AAC ATC NI

Findings

EcoRl cleaved