TY - JOUR
T1 - Structure-guided functional characterization of enediyne self-sacrifice resistance proteins, CalU16 and CalU19
AU - Elshahawi, Sherif I.
AU - Ramelot, Theresa A.
AU - Seetharaman, Jayaraman
AU - Chen, Jing
AU - Singh, Shanteri
AU - Yang, Yunhuang
AU - Pederson, Kari
AU - Kharel, Madan K.
AU - Xiao, Rong
AU - Lew, Scott
AU - Yennamalli, Ragothaman M.
AU - Miller, Mitchell D.
AU - Wang, Fengbin
AU - Tong, Liang
AU - Montelione, Gaetano T.
AU - Kennedy, Michael A.
AU - Bingman, Craig A.
AU - Zhu, Haining
AU - Phillips, George N.
AU - Thorson, Jon S.
N1 - Publisher Copyright:
© 2014 American Chemical Society.
PY - 2014/10/17
Y1 - 2014/10/17
N2 - 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 1 in 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.
AB - 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 1 in 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.
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U2 - 10.1021/cb500327m
DO - 10.1021/cb500327m
M3 - Article
C2 - 25079510
AN - SCOPUS:84908146558
SN - 1554-8929
VL - 9
SP - 2347
EP - 2358
JO - ACS Chemical Biology
JF - ACS Chemical Biology
IS - 10
ER -