Calicheamicin γ1I (1)
is an enediyne antitumor compound produced by Micromonospora
echinospora spp. calichensis, and its biosynthetic gene cluster
has been previously reported. Despite extensive analysis and biochemical
study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach
and biochemical characterization, two proteins encoded by genes from
the 1 biosynthetic gene cluster assigned as “unknowns”,
CalU16 and CalU19, were characterized. Structure analysis revealed
that they possess the STeroidogenic Acute Regulatory protein related
lipid Transfer (START) domain known mainly to bind and transport lipids
and previously identified as the structural signature of the enediyne
self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were
cleaved by 1in vitro. Through site-directed
mutagenesis and mass spectrometry, we identified the site of cleavage
in each protein and characterized their function in conferring resistance
against 1. This report emphasizes the importance of structural
genomics as a powerful tool for the functional annotation of unknown
proteins.