Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli.
The methods of homologous high-level expression and simple large-scale purification for coenzyme B(12)-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 alpha(6)beta(6). Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B(12), the binding of 1 mol of the inhibitor per mol of the alphabeta 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.
Akita
Keita
K
Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
Hieda
Naoki
N
Baba
Nobuyuki
N
Kawaguchi
Satoshi
S
Sakamoto
Hirohisa
H
Nakanishi
Yuka
Y
Yamanishi
Mamoru
M
Mori
Koichi
K
Toraya
Tetsuo
T
D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D004981:Ethanolamine Ammonia-Lyase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D011994:Recombinant Proteins; D004981:Ethanolamine Ammonia-Lyase