L-Lysine cyclodeaminase from Streptomyces pristinaespiralis was heterologously expressed in Escherichia coli, isolated to 90% purity after two purification steps and characterised. The size of the isolated recombinant enzyme was in agreement with the theoretical size calculated from the corresponding gene. We demonstrated that our preparation converts L-lysine to L-pipecolic acid (enantiomeric excess >95%) after isolating and identifying the conversion product by LC/MS, NMR and IR. This conversion followed Michaelis-Menten kinetics with a K(m) of 1.39+/-0.32 mM. The enzyme activity was maximal at pH 6.7. Reducing conditions, the presence of glycerol and in particular the presence of iron(II) significantly enhanced the L-lysine cyclodeaminase activity. Although the heat stability of the enzyme diminished significantly after 37 degrees C, the initial rate of reaction was maximal at 61 degrees C. We found no requirement for an external cofactor for full activity, although sequence data indicate NAD+ as cofactor. Upon enzyme denaturation, NAD+ release was observed, which indicates very tight binding of NAD+ to the enzyme. In parallel we developed selection and screening assays for lysine cyclodeaminase, which we adapted to microtitre plate format and validated. Among twenty-eight lysine analogues screened for turnover/binding to the enzyme, three were identified as substrates (L-ornithine, 5-hydroxylysine and L-4-thialysine), while another six (4-azalysine, L-2,4-diaminobutyric acid, 1,5-diaminopentane, N-epsilon-trifluoroacetyl-L-lysine, N-epsilon-Boc-L-lysine and N-epsilon-methyl-L-lysine) were shown to compete against L-lysine turnover without being converted by the enzyme. All substrates displayed Michaelis-Menten kinetics upon turnover by lysine cyclodeaminase. Our results indicate that the lysine cyclodeaminase from Streptomyces pristinaespiralis is a highly enantioselective enzyme at the substrate recognition and conversion levels, in both cases in favour of the l-isomer.