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

MOLECULAR CHARACTERIZATION OF NATIVE AND RECOMBINANT APOLIPOPROTEIN A-I-MILANO DIMER - THE INTRODUCTION OF AN INTERCHAIN DISULFIDE BRIDGE REMARKABLY ALTERS THE PHYSICOCHEMICAL PROPERTIES OF APOLIPOPROTEIN-A-I

The disulfide-linked dimer of apolipoprotein A-IMilano (A-IM/A-IM), a natural Arg173 \u2192 Cys variant of apoA-I, was purified from carriers' plasma and produced in Escherichia coli. The recombinant A-IM/A-IM is identical to native A-IM/A-IM, by mass spectrometry, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing. Lipid-free A-IM/A-IM undergoes concentration-dependent self-association similar to apoA-I, but at all concentrations apoA-I is more self-associated than A-IM/A-IM. Far-ultraviolet CD spectra of A-IM/A-IM reveal a highly \u3b1-helical structure predicted to be 3c65% in the lipid-free and 3c78% in the lipid-associated states, versus 43 and 73% for apoA-I. A significant loss of \u3b1-helix occurs below pH 3.5 and above pH 10 in both apoA-I and A-IM/A-IM; A-IM/A-IM constantly shows a higher \u3b1-helical content than apoA-I over the entire pH range (1.7-12.8), suggesting that hydrophobic forces stabilize the interaction between the two A-IM chains. Indeed, and differently from apoA-I, the \u3b1-helical content of A-IM/A-IM is minimally affected by solvent ionic strength. The aromatic side chains in both lipid-free and lipid-bound A-IM/A-IM are immobilized in a more asymmetric and hydrophobic environment than in lipid-free apoA-I, the conformation of A-IM/A-IM being instead similar to that achieved by apoA-I following interaction with lipids. The present findings prove that rA-IM/A-IM is structurally identical to the native protein; the conformation of A-IM/A-IM is remarkably different from that of apoA-I, thus possibly explaining some of the peculiar functional properties of the apoA-IMilano dimer