Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli.
A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.
Kälin
M
M
Institute for Biotechnology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich.
Neujahr
H Y
HY
Weissmahr
R N
RN
Sejlitz
T
T
Jöhl
R
R
Fiechter
A
A
Reiser
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D005800:Genes, Fungal; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014250:Trichosporon / Q000201:enzymology* / Q000235:genetics*
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase