Abstract
A gene encoding an L-lysine dehydrogenase was identified in the hyperthermophilic archaeon Pyrococcus horikoshii. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme is the most thermostable L-lysine dehydrogenase yet described, with a half-life of 180 min at 100 degrees C. The product of the enzyme's catalytic activity is Delta(1)-piperideine-6-carboxylate, which makes this enzyme an L-lysine 6-dehydrogenase (EC 1.4.1.18) that catalyzes the reductive deamination of the epsilon- amino group and a type of NAD-dependent amine dehydrogenase. The three-dimensional structure of the enzyme was determined using the mercury-based multiple-wavelength anomalous dispersion method at a resolution of 2.44 A in the presence of NAD and sulfate ion. The asymmetric unit consisted of two subunits, and a crystallographic 2-fold axis generated the functional dimer. Each monomer consisted of a Rossmann fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed similarity to that of saccharopine reductase. Notably, the structures of subunits A and B differed significantly. In subunit A, the active site contained a sulfate ion that was not seen in subunit B. Consequently, subunit A adopted a closed conformation, whereas subunit B adopted an open one. In each subunit, one NAD molecule was bound to the active site in an anti-conformation, indicating that the enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH (type A specificity). This is the first description of the three-dimensional structure of l-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase.
Highlights
The gene encoding LysDH in Geobacillus stearothermophilus was cloned and expressed in Escherichia coli, after which the primary structure of the enzyme was determined (2)
LysDH is regarded as a type of NAD(P)-dependent amine dehydrogenase because it catalyzes the dehydrogenation at the ⑀-carbon with deamination, whereas other amino acid dehydrogenases generally catalyze the oxidative deamination of the ␣-amino group to form the corresponding keto acid
Within the genomic sequence of an anaerobic hyperthermophilic archaeon, Pyrococcus horikoshii, we found a gene whose predicted amino acid sequence exhibits 33% identity to that of droxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; multiple-wavelength anomalous dispersion (MAD), multiplewavelength anomalous dispersion
Summary
Purification from Inclusion Bodies—To purify LysDH from inclusion bodies, E. coli transformants were collected, suspended in 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM -mercaptoethanol and 4% Triton X-100, and disrupted by sonication. Refolded LysDH was heated at 80 °C for 10 min and clarified by centrifugation, after which the protein in the supernatant was run on a Superdex 200 column (Amersham Biosciences) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM -mercaptoethanol and 200 mM NaCl, and the resultant protein solution was used for enzymological characterization. PH Optimum, and Kinetic Parameters—To determine the effect of temperature on its stability, the enzyme was incubated at different temperatures in 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM -mercaptoethanol.
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