Cytochrome c peroxidase from a methylotrophic yeast: physiological role and isolation

Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (car) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source, A large collection of second-site suppressor catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth(+)) in the absence of catalase activity. These Mth(+) Ro I car scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wildtype strain. In contrast to the parental C-105 strain, H2O2 does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor strains is strongly correlated with increased levels of the alternative H2O2-destroying enzyme, cytochrome c peroxidase, Cytochrome c peroxidase from cell-free extracts of one of the scn revertants has been purified to homogeneity and crystallized.