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b-Lysine 5,6-Aminomutase (D-Lysine 5,6-Aminomutase)

Enzyme Properties An enzyme complex containing a ¡-lysine 5,6-aminomutase (EC 5.4.3.3) and a D-lysine 5,6-aminomutase (EC 5.4.3.4) has been isolated from Clostridium sticklandii. The enzyme was purified as a single protein complex that accepts two different substrates, both ^¡-lysine and D-lysine. The products of the mutase reaction are (3S,5S)-diaminohexanoic acid 45 and (2R,5S)-diaminohexanoic acid, respectively. Approximately 35 years ago the enzyme was discovered by Stadt-man and coworkers [68]. Recently the two genes encoding the aminomutase have been cloned, sequenced, and heterologously expressed in E. coli [69]. The enzyme is a 170 kD complex consisting of two 55 kDa and 30 two kDa subunits. The aminomutase is stimulated by B12 and pyridoxal phosphate. A rapid turnover-associated inactivation occurs both for the enzyme purified from C. sticklandii and in the recombinant protein. Analysis of the 5,6-aminomutase amino acid sequence reveals a region in the small subunit that has similarities with other cobalt-dependent mutases probably responsible for the binding of B12 [69]. In the d-lysine 5,6-aminomutase from Porphyromonas gingivalis an essential lysine residue was detected that is part of a PLP binding motif [70].

Stereochemical Aspects The configuration of the product of the ¡-lysine 2,3-aminomutase reaction was established to be 3S,5S by NMR spectroscopic comparison of the ¿-lactam of the 3,5-diaminohexanoic acid 45 from C. sticklandii with authentic samples of both pairs of diastereomers [71]. The stereochemical mechanism of the ¡-lysine 5,6-aminomutase reaction was investigated by means of tritium labeling. It turned out that the pro-5S hydrogen atom migrates to position 6 in the 3,5-diaminohexanoic acid [72] (Scheme 1.6.16).

Scheme 1.6.16. Biosynthesis of (3S,5S)-diaminohexanoic acid in Clostridium sticklandii.

Reaction Mechanism Although the ¡-lysine 5,6-aminomutase requires cobalamin as cofactor instead of SAM its reaction mechanism seems to be similar to that of the lysine 2,3-aminomutase [73]. Experiments with tritium-labeled lysine and B12 showed that B12 is directly involved in the hydrogen shift from position 5 in D-

lysine to position 6 in 2,5-diaminohexanoic acid [74]. Also PLP is required for enzyme activity [75]. The task of PLP in the mutase reaction is recognized to stabilize the radical intermediates by imine formation [76, 77].

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