FAD and substrate analogs as probes for lysine N6-hydroxylase from Escherichia coli EN 222.

Abstract

Lysine N6-hydroxylase catalyzes the hydroxylation of the N-terminal amino function of L-lysine at the expense of NADPH and molecular oxygen. The enzyme also requires FAD for its catalytic activity. Unlike other flavoprotein monooxygenases, binding of FAD is rather weak with a Kd of 30 microM at 4 degrees C. The spectral properties of FAD bound to lysine N6-hydroxylase are very similar to free oxidized FAD. In the absence of substrate, the enzyme has an NADPH oxidase activity which results in the generation of hydrogen peroxide. With increasing concentration of L-lysine, the NADPH oxidase activity is enhanced up to 10-fold and the generation of hydrogen peroxide decreases. At the same time, the substrate is hydroxylated. Km values for L-lysine and FAD were determined as 105 microM and 0.7 microM, respectively. Utilizing FAD analogs, we could demonstrate that L-lysine exerts its effector role mostly on the reductive half reaction of the overall catalytic cycle. Prolonged incubation of the enzyme with either 8-chloro- or 8-fluoro-FAD gave rise to a covalently attached flavin which is formed as a result of the nucleophilic attack of a thiolate on the 8-position of the flavin. Several lines of evidence indicate that the reaction takes place in the FAD binding site of the protein. The substrate specificity was investigated using amino acids with various lengths of side chain. L-Lysine and derivatives with similar side chain length are hydroxylated by lysine N6-hydroxylase. Ornithine, the lower homolog of lysine, was not hydroxylated and did not affect the NADPH oxidase activity of the enzyme. On the other hand, homolysine accelerated the rate of NADPH oxidation but was not hydroxylated. Additional requirements for efficient hydroxylation were also investigated using a variety of substrate analogs. From these studies a schematic structure of the active site of the enzyme was deduced. Sequence comparison of the FAD binding site of various flavoproteins revealed possible factors for weak binding of the cofactor in the case of lysine N6-hydroxylase.