Lanthionine synthase from the oral bacterium Fusobacterium nucleatum is a fold type II pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine by a second molecule of l-cysteine to form H2S and l-lanthionine. The meso-isomer of the latter product is incorporated into the F. nucleatum peptidoglycan layer. Herein, we investigated the catalytic role of S224, which engages in hydrogen-bond contact with the terminal carboxylate of l-lanthionine in the closed conformation of the enzyme. Unexpectedly, the S224A variant elicited a 7-fold increase in the turnover rate for H2S and lanthionine formation and a 70-fold faster rate constant for the formation of the α-aminoacrylate intermediate compared to the wild-type enzyme. Presteady state kinetic analysis further showed that the reaction between S224A and l-cysteine leads to the formation of the more reactive ketoenamine tautomer of the α-aminoacrylate. The α-aminoacrylate with the protonated Schiff base is not an observable intermediate in the analogous reaction with the wild type, which may account for its attenuated kinetic properties. However, the S224A substitution is detrimental to other aspects of the catalytic cycle; it facilitates the α,β-elimination of l-lanthionine, and it weakens the enzyme's catalytic preference for the formation of l-lanthionine over that of l-cystathionine.