Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli

Keita Akita, Naoki Hieda, Nobuyuki Baba, Satoshi Kawaguchi, Hirohisa Sakamoto, Yuka Nakanishi, Mamoru Yamanishi, Koichi Mori, Tetsuo Toraya

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The methods of homologous high-level expression and simple large-scale purification for coenzyme B12-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer α6β6. Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B12, the binding of 1 mol of the inhibitor per mol of the αβ unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme.

Original languageEnglish
Pages (from-to)83-93
Number of pages11
JournalJournal of Biochemistry
Volume147
Issue number1
DOIs
Publication statusPublished - Jan 2010

Fingerprint

Ethanolamine Ammonia-Lyase
Escherichia coli
Purification
Enzymes
Catalysis
Chloromercuribenzoates
Agglomeration
Enzyme Stability
Proteolysis
Vitamin B 12
Operon
Cobalt
Detergents
Solubility
Trypsin
Titration
Catalytic Domain
Paramagnetic resonance

Keywords

  • Adenosylcobalamin
  • Coenzyme B
  • Ethanolamine ammonia-lyase
  • Radical enzyme
  • Truncated enzyme

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., ... Toraya, T. (2010). Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli. Journal of Biochemistry, 147(1), 83-93. https://doi.org/10.1093/jb/mvp145

Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli. / Akita, Keita; Hieda, Naoki; Baba, Nobuyuki; Kawaguchi, Satoshi; Sakamoto, Hirohisa; Nakanishi, Yuka; Yamanishi, Mamoru; Mori, Koichi; Toraya, Tetsuo.

In: Journal of Biochemistry, Vol. 147, No. 1, 01.2010, p. 83-93.

Research output: Contribution to journalArticle

Akita, K, Hieda, N, Baba, N, Kawaguchi, S, Sakamoto, H, Nakanishi, Y, Yamanishi, M, Mori, K & Toraya, T 2010, 'Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli', Journal of Biochemistry, vol. 147, no. 1, pp. 83-93. https://doi.org/10.1093/jb/mvp145
Akita, Keita ; Hieda, Naoki ; Baba, Nobuyuki ; Kawaguchi, Satoshi ; Sakamoto, Hirohisa ; Nakanishi, Yuka ; Yamanishi, Mamoru ; Mori, Koichi ; Toraya, Tetsuo. / Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli. In: Journal of Biochemistry. 2010 ; Vol. 147, No. 1. pp. 83-93.
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