The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

Yousef I. Hassan, Hideaki Moriyama, Janos Zempleni

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′-AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site-directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed.

Original languageEnglish (US)
Pages (from-to)35-41
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume495
Issue number1
DOIs
StatePublished - Mar 1 2010

Fingerprint

Biotinylation
Peptides
Lysine
Adenosine Monophosphate
Histones
Arginine
Assays
Proteolysis
Mutagenesis
Two-Hybrid System Techniques
Biotin
Site-Directed Mutagenesis
holocarboxylase synthetases
Computer Simulation
Yeast
Glycine
Catalytic Domain
Proteins
Substitution reactions

Keywords

  • Biotin
  • Domains
  • Holocarboxylase synthetase
  • Substrate
  • Syn67

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

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title = "The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation",
abstract = "Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′-AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site-directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed.",
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author = "Hassan, {Yousef I.} and Hideaki Moriyama and Janos Zempleni",
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TY - JOUR

T1 - The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

AU - Hassan, Yousef I.

AU - Moriyama, Hideaki

AU - Zempleni, Janos

PY - 2010/3/1

Y1 - 2010/3/1

N2 - Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′-AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site-directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed.

AB - Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′-AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site-directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed.

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KW - Domains

KW - Holocarboxylase synthetase

KW - Substrate

KW - Syn67

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