The yodO gene product of Bacillus subtilis has been cloned and overexpressed in Escherichia coli and purified. The nucleotide sequence encodes a protein of 471 amino acids with a calculated molecular mass of 54071 Da. The translated amino acid sequence is more than 60% identical to that of the lysine 2,3-aminomutase from Clostridium subterminale SB4. Analytical HPLC gel-permeation chromatography leads to an estimate of an overall molecular mass of 224000±21000 Da, which corresponds to a tetrameric protein. The purified protein contains iron, sulphide and pyridoxal 5ʹ-phosphate (PLP) and displays an optical absorption band extending to 700 nm, suggesting the presence of an iron-sulphide cluster. After reductive incubation with L-cysteine anaerobically, the protein catalyses the transformation of L-lysine into β-lysine in the presence of S-adenosylmethionine (AdoMet) and sodium dithionite. The Km value for L-lysine is estimated to be 8.0±2.2 mM. The iron-sulphur centre is stable in air, allowing aerobic purification. EPR spectroscopy at 10 K of the purified enzyme revealed an EPR signal similar to that of the [4Fe-4S]3+ cluster observed in the clostridial lysine 2,3-aminomutase. Incubation with cysteine under anaerobic conditions converts the iron-sulphur centre into the EPR-silent [4Fe-4S]2+. Unlike the clostridial enzyme, the fully reduced [4Fe-4S]+ could not be characterized by further reduction with dithionite in the presence of AdoMet, although both dithionite and AdoMet were required to activate the enzyme. Upon addition of L-lysine, dithionite and AdoMet to the reduced enzyme and freezing the solution to 77 K, the EPR spectrum revealed the presence of an organic free-radical signal (g = 2.0023), which displayed multiple hyperfine transitions very similar to the spectrum of the β-lysine-related radical in the mechanism of the clostridial lysine 2,3-aminomutase. Experiments with isotopically substituted L-lysine and lysine analogues verified the association of spin density with the carbon skeleton of lysine. The data indicate that the protein encoded by the yodO gene of B. subtilis is a novel lysine 2,3-aminomutase. The E. coli homologue of clostridial lysine 2,3-aminomutase was also expressed in E. coli and purified. This protein contained iron and sulphide but not PLP, it did not display lysine 2,3-aminomutase activity, and addition of PLP did not induce 2,3-aminomutase activity.

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