Catalytic mechanism of bleomycin N-acetyltransferase proposed on the basis of its crystal structure.
Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA(2), and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism.
Oda
Kosuke
K
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Matoba
Yasuyuki
Y
Noda
Masafumi
M
Kumagai
Takanori
T
Sugiyama
Masanori
M
D000105:Acetyl Coenzyme A / Q000737:chemistry*; D000123:Acetyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001761:Bleomycin / Q000737:chemistry*; D002384:Catalysis; D018360:Crystallography, X-Ray; D017434:Protein Structure, Tertiary / Q000502:physiology; D013302:Streptomyces / Q000201:enzymology*
D001426:Bacterial Proteins; D001761:Bleomycin; D000105:Acetyl Coenzyme A; C087954:bleomycin N-acetyltransferase; D000123:Acetyltransferases