N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition

Yousef I. Hassan, Hideaki Moriyama, Lars J. Olsen, Xin Bi, Janos Zempleni

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)183-188
Number of pages6
JournalMolecular Genetics and Metabolism
Volume96
Issue number4
DOIs
StatePublished - Apr 1 2009

Fingerprint

Substrates
Methylmalonyl-CoA Decarboxylase
Biotin
Yeast
Assays
biotin carboxylase
Metabolism
Adenosine Triphosphate
Carboxylation
Biotinylation
holocarboxylase synthetases
Two-Hybrid System Techniques
Computational Biology
Catalysis
Vitamins
Computer Simulation
Histones
Saccharomyces cerevisiae
Fatty Acids
Yeasts

Keywords

  • BirA
  • Domains
  • Holocarboxylase synthetase
  • Propionyl-CoA carboxylase
  • p67

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Endocrinology

Cite this

N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition. / Hassan, Yousef I.; Moriyama, Hideaki; Olsen, Lars J.; Bi, Xin; Zempleni, Janos.

In: Molecular Genetics and Metabolism, Vol. 96, No. 4, 01.04.2009, p. 183-188.

Research output: Contribution to journalArticle

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abstract = "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.",
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