TY - JOUR
T1 - N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition
AU - Hassan, Yousef I.
AU - Moriyama, Hideaki
AU - Olsen, Lars J.
AU - Bi, Xin
AU - Zempleni, Janos
N1 - Funding Information:
A contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. Additional support was provided by NIH Grants DK063945, DK077816, GM62484 and ES015206, USDA Grant 2006-35200-17138, and by NSF grants EPS-0701892 and MCB6552870.
PY - 2009/4
Y1 - 2009/4
N2 - Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP-binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS.
AB - Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP-binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS.
KW - BirA
KW - Domains
KW - Holocarboxylase synthetase
KW - Propionyl-CoA carboxylase
KW - p67
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U2 - 10.1016/j.ymgme.2008.12.006
DO - 10.1016/j.ymgme.2008.12.006
M3 - Article
C2 - 19157941
AN - SCOPUS:61849137804
VL - 96
SP - 183
EP - 188
JO - Molecular Genetics and Metabolism
JF - Molecular Genetics and Metabolism
SN - 1096-7192
IS - 4
ER -