The
bleomycin-inactivating
enzyme,
bleomycin hydrolase, is believed to be involved in
tumor resistance to the anticancer
drug bleomycin. This homohexamer is an
aminopeptidase that shows homology to
cysteine proteinases around the
cysteine and
histidine active site. The role that these residues play in hydrolyzing
bleomycin and in hexamer oligomerization of
bleomycin hydrolase is not known. In this study, the yeast
bleomycin hydrolase gene was expressed in Escherichia coli, and site-directed mutagenesis was employed to precisely investigate the roles of the conserved Cys102 and His398 residues in its structure and enzymatic activity. Three mutants were created, in which Cys102 was replaced by
arginine or
serine, and His398 was changed to
glycine. The ability of
bleomycin hydrolase to oligomerize was neither affected by the subtle
cysteine/
serine mutation nor affected by
cysteine/
arginine or
histidine/
glycine mutations. However, the ability of
bleomycin hydrolase to hydrolyze and inactivate
bleomycin was totally abolished in all three mutants, suggesting that the
cysteine thiol and
histidine imidazole are critical for hydrolyzing
bleomycin. Furthermore, in contrast to predictions from the recently reported crystal structure of this
enzyme, hexamer formation is not required for the enzymatic activity of
bleomycin hydrolase. Thus, these results demonstrate that Cys102 and His398 are required for
bleomycin hydrolase activity but not hexamer formation, and that both monomer and hexamer are active forms of
bleomycin hydrolase.