SOURCES AND NATURE OF HETEROGENEITY IN RECOMBINANT PHENOL HYDROXYLASEDERIVED FROM THE BASIDIOMYCETOUS SOIL YEAST TRICHOSPORON-CUTANEUM

Preparations of the dimeric flavoenzyme phenol hydroxylase derived fro m Trichosporon cutaneum were found to contain an active tetrameric for m when the enzyme was produced in Escherichia coil, The relative conte nt of the tetramer was estimated from scans of silver-stained native P AGE 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 ex tracts or purified preparations from T. cutaneum, Traces of higher mul timers and of possibly deamidated species were also detected in prepar ations 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 fermenter culture appears to be of mi nor importance, The dimeric and the tetrameric forms were purified usi ng either SEC or ion-exchange chromatography as a final step, The two purified species did not interchange under a variety of conditions, in dicating that they are not undergoing rapid equilibria. The FAD of eit her form, as isolated by SEC, was present to a lower-than-expected ext ent of 2 equiv./dimer. However, by removing FAD and reconstituting the resulting apoproteins with the cofactor, the FAD content could be inc reased to 2 equiv, in the dimer and 3 equiv, in the tetramer, Both rec onstituted forms exhibited absorption spectra identical with that of p henol hydroxylase from T. cotaneum 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 dime ric and the tetrameric forms was, however, lower than expected from th e ratio of their FAD contents. The results are compatible with the not ion that the tetramer consists of a native phenol hydroxylase dimer as sociated with a non-native one with a decreased ability to bind FAD, e ither in one or both of its constituent 'monomers'.