title: Sources and nature of heterogeneity in recombinant phenol hydroxylase derived from the basidiomycetous soil yeast Trichosporon cutaneum.
abstract: Preparations of the dimeric flavoenzyme phenol hydroxylase derived from Trichosporon cutaneum were found to contain an active tetrameric form when the enzyme was produced in Escherichia coli. The relative content of the tetramer was estimated from scans of silver-stained native PAGE gels and/or size-exclusion chromatography (SEC). Proportions of up to 22% of the enzyme protein, depending on the growth temperature and the level of added inducer, were observed in independent cultures as well as in purified preparations. No tetramer was ever seen in cell extracts or purified preparations from T. cutaneum. Traces of higher multimers and of possibly deamidated species were also detected in preparations of the recombinant enzyme. The rate of enzyme production seems to be the major factor in promoting formation of the tetramer, whereas the specific growth rate of the fermentor culture appears to be of minor importance. The dimeric and the tetrameric forms were purified using either SEC or ion-exchange chromatography as a final step. The two purified species did not interchange under a variety of conditions, indicating that they are not undergoing rapid equilibria. The FAD of either form, as isolated by SEC, was present to a lower-than-expected extent of 2 equiv/dimer. However, by removing FAD and reconstituting the resulting apoproteins with the cofactor, the FAD content could be increased to 2 equiv. in the dimer and 3 equiv. in the tetramer. Both reconstituted forms exhibited absorption spectra identical with that of phenol hydroxylase from T. cutaneum as well as that of the recombinant enzyme. All spectra were equally perturbed by one equivalent of phenol per enzyme-attached FAD. The ratio of specific activities of the dimeric and the tetrameric forms was, however, lower than expected from the ratio of their FAD contents. The results are compatible with the notion that the tetramer consists of a native phenol hydroxylase dimer associated with a non-native one with a decreased ability to bind FAD, either in one or both of its constituent 'monomers'.
authors.affiliation: Department of Biochemistry and Biochemical Technology, Kungliga Tekniska Högskolan, Stockholm, Sweden.
authors.forename: S
authors.initials: S
authors.lastname: Waters
authors.forename: H Y
authors.initials: HY
authors.lastname: Neujahr
mesh_terms: D001665:Binding Sites; D002455:Cell Division / Q000235:genetics; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011108:Polymers; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016622:Silver Staining; D013696:Temperature; D014250:Trichosporon / Q000166:cytology / Q000201:enzymology*
chemical_list: D003470:Culture Media; D011108:Polymers; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase