Expression, crystallization and preliminary X-ray crystallographic study of ethanolamine ammonia-lyase fromEscherichia coli

Abstract

Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. The wild-type enzyme shows a very low solubility. N-terminal truncation of the Escherichia coli EAL beta-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties. Two deletion mutants of the enzyme [EAL(betaDelta4-30) and EAL(betaDelta4-43)] have been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method. Crystals of EAL(betaDelta4-30) and EAL(betaDelta4-43) diffracted to approximately 8.0 and 2.1 A resolution, respectively.

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12‐Dependent Diol Dehydratase and Ethanolamine Ammonia‐Lyase

“…Expression and purification of EAL : Expression and sample preparation of EAL(Δβ4–43), which contains the full‐length α‐subunit and the His 6 ‐tagged β‐subunit lacking the residues Lysβ4–Cysβ43, were carried out by a similar method to that for DD except that the cell lysate was bound to Ni‐NTA and eluted with an elution buffer containing 250 mM imidazole without removing the His 6 ‐tag [7c,30] . The purified sample was dialyzed against 20 mM Tris⋅HCl (pH 8.0), 0.2 mM EA (pH 8.0), and 1 mM dithiothreitol, followed by incubation at 30 °C for 30 min in the presence of trace AdoCbl (molar ratio 1 : 50 to apoenzyme).…”

How Coenzyme B12-Dependent Ethanolamine Ammonia-Lyase Deals with Both Enantiomers of 2-Amino-1-propanol as Substrates: Structure-Based Rationalization,,

“…Escherichia coli JM109 cells carrying pUSI2ENd(EAL(Δβ4-43)) plasmid were used for homologous overexpression of E. coli EAL(βΔ4-43) which consists of the R subunit and the N-terminal His6-tagged β subunit lacking residues Lysβ4-Cysβ43 (27). Purification of EAL(βΔ4-43) was achieved via affinity chromatography on Ni-NTA agarose (Qiagen) and size-exclusion chromatography, as described previously (27). The final protein solution contained 20 mg/mL enzyme, 10 mM Tris-HCl buffer (pH 8.0) containing 200 mM KCl, 1 mM dithiothreitol (DTT), and 20 μM CN-Cbl.…”

“…Crystals of the EAL(Δβ4−43)−CN-Cbl complex bound to ( R )- or ( S )-2-amino-1-propanol were prepared by the following procedure. Briefly, crystals of the substrate-free form of the EAL(Δβ4−43)−CN-Cbl complex were obtained by the sitting drop vapor diffusion method using a well solution containing 6.0−7.0% (w/v) polyethylene glycol 4000, 24−26% (v/v) glycerol, 1.0% (v/v) 2-methyl-2,4-pentanediol (MPD), and 0.1 M imidazole-HCl (pH 6.3), as described previously . Thus obtained substrate-free crystals were harvested and soaked for 30 min in the mother liquor supplemented with 10 mM ( R )- or ( S )-2-amino-1-propanol (pH 8.0) as well as each component of the substrate-free final buffer.…”