Identification of a novel pyridoxal 5'-phosphate binding site in adenosylcobalamin-dependent lysine 5,6-aminomutase from Porphyromonas gingivalis.

Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversion of D-lysine with 2,5-diaminohexanoate and of L-beta-lysine with 3,5-diaminohexanoate. The coenzymes for 5,6-LAM are adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP). In the proposed chemical mechanism, AdoCbl initiates the formation of substrate radicals, and PLP facilitates the radical rearrangement by forming an external aldimine linkage with the epsilon-amino group of a substrate, either D-lysine or L-beta-lysine. In the resting enzyme, an internal aldimine between PLP and an essential lysine in the active site facilitates productive PLP binding and catalysis. We present here biochemical, biophysical, and site-directed mutagenesis experiments, which document the existence of an essential lysine residue in the active site of 5,6-LAM from Porphyromonas gingivalis. Reduction of 5,6-LAM with NaBH(4) rapidly inactivates the enzyme and shifts the electronic absorption band from 420 to 325 nm. This is characteristic of the reduction of an aldimine linkage between the carbonyl group of PLP and the epsilon-amino group of a lysine residue. The reduced peptide was identified by Q-TOF/MS and further confirmed by Q-TOF/MS/MS sequencing. We show that lysine 144 in the small subunit of 5,6-LAM is the essential lysine residue. Lysine 144(beta) is separated by only 11 amino acids from histidine 133(beta), which forms a part of the "base-off"-AdoCbl binding motif. The sequence of the novel PLP-binding motif is conserved in 5,6-LAM from Clostridium sticklandii and P. gingivalis, and it is distinct from all known PLP-binding motifs. Mutation of lysine 144(beta) to glutamine led to K144Q(beta)-5,6-LAM, which displayed no enzymatic activity and no absorption band corresponding to an internal PLP-aldamine. In summary, we introduce a novel PLP-binding motif, the first to be discovered in an AdoCbl-dependent enzyme.