Biosynthesis of meso-lanthionine in Fusobacterium nucleatum

Abstract

The opportunistic oral pathogen, Fusobacterium nucleatum contains meso-lanthionine as the diaminodicarboxylic acid in the pentapeptide crosslink of the peptidoglycan layer. The diastereomer, l,l-lanthionine is formed by lanthionine synthase, a PLP-dependent enzyme that catalyzes the β-replacement of l-cysteine with a second equivalent of l-cysteine. In this study, we explored possible enzymatic mechanisms for the formation of meso-lanthionine. Our inhibition studies with lanthionine synthase, described herein, revealed that meso-diaminopimelate, a bioisostere of meso-lanthionine, is a more potent inhibitor of lanthionine synthase compared to the diastereomer, l,l-diaminopimelate. These results suggested that lanthionine synthase could also form meso-lanthionine by the β-replacement of l-cysteine with d-cysteine. Through steady-state and pre-steady state kinetic analysis, we confirm that d-cysteine reacts with the ⍺-aminoacylate intermediate with a kon that was 2-3-fold faster and Kd value that was 2–3fold lower compared to l-cysteine. However, given that intracellular levels of d-cysteine levels are assumed to be significantly lower than that of l-cysteine, we also determined if the gene product, FN1732, with low sequence identity to diaminopimelate epimerase could convert l,l-lanthionine to meso-lanthionine. Using diaminopimelate dehydrogenase in a coupled spectrophotometric assay, we show that FN1732 can convert l,l-lanthionine to meso-lanthionine with a kcat of 0.07 ± 0.001 s−1 and a KM of 1.9 ± 0.1 mM. In summary, our results provide two possible enzymatic mechanisms for the biosynthesis of meso-lanthionine in F. nucleatum.