Sodium iminodisuccinate (IDS) is a widely used synthetic, medium-strong chelator belonging to the group of aminopolycarboxylates. IDS can be used as a substitute for EDTA, which is not biodegradable according to OECD tests 301A, 301F, and 302B (19). In contrast, all three epimers of IDS (R,S-, S,S-, and R,R-IDS), are readily biodegradable (9).The bacterial strains Ralstonia sp. strain SLRS7 and Agrobacterium tumefaciens BY6 grow on IDS as the sole source of carbon and nitrogen. Cofactor-independent C-N lyases catalyze the cleavage of IDS, generating D-aspartic acid and fumaric acid from R,S-IDS as well as L-aspartic acid and fumaric acid from S,S-IDS (9, 10). R,R-IDS metabolism is preceded by epimerization. A cofactor-independent epimerase from A. tumefaciens BY6 catalyzes the conversion of all three epimers into each other (9). For the lyases and the epimerase, the natural function is still unknown.Here, we report sequences, cloning, recombinant expression, and purification of the IDS-converting enzymes. MATERIALS AND METHODSStrains, plasmids, and growth conditions. The isolation and growth conditions of A. tumefaciens BY6 and of Ralstonia sp. strain SLRS7 have been reported previously (9, 10). Escherichia coli strains were grown aerobically in LuriaBertani (LB) or 2ϫ tryptone-yeast extract medium (1) supplemented with final concentrations of 100 g ml Ϫ1 ampicillin, 25 g ml Ϫ1 chloramphenicol, and 50 g ml Ϫ1 kanamycin according to the requirements. A list of strains and plasmids used and constructed is given in Table 1.Protein purification from wild-type strains. The purification of IDS-epimerase and IDS-lyase has been described previously (9, 10). IDS-lyase from Ralstonia sp. strain SLRS7 was purified with slight modifications of the original protocol. An initial (NH 4 ) 2 SO 4 (40% [wt/vol]) precipitation of the lyase was applied. As an additional purification step, hydroxyapatite chromatography was performed with a Bio-Scale cHT5-1 (Bio-Rad Laboratories, Germany) column, using a linear gradient comprising 2.5 mM potassium phosphate, pH 7.5 (buffer A), and 500 mM potassium phosphate, pH 7.5 (buffer B), with ϩ0.4% (vol/vol) buffer B per ml. Elution of IDS-lyase started with 12% (vol/vol) buffer B. A specific activity of up to 10 mol IDS min Ϫ1 mg Ϫ1 was obtained for SLRS7-lyase.Protein cleavage, peptide separation, and sequencing. Trypsin digestion of 200 g protein was based on the method of Stone et al. (24). Separation of tryptic peptides of epimerase and SLRS7-lyase by reversed-phase high-pressure liquid chromatography was performed using a 250-by 4-mm Grom-Sil ODS-5 column (where ODS is octyldecyl silane) (Grom, Germany) and an aqueous solution of 0.1% (vol/vol) trifluoroacetic acid (solvent A) and 0.085% (vol/vol) trifluoroacetic acid in 80% (vol/vol) acetonitrile (solvent B). The following gradients were applied: 0 to 7.5 ml, 7% B; 7.5 to 57.5 ml, 7 to 50% B; and 57.5 to 77.5 ml, 50 to 98% B (0.5 ml min Ϫ1 ; detection at 210 nm). The BY6-lyase fragments were fractionated using a 100-by 2-mm TSK-Gel Super...
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