POA was found to be directly tethered to a divalent metal cation. In a more recent publication, French et al. reported several high-resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae in unliganded and ligand-bound forms, even with a trapped nicotinoyl thioester intermediates. These crystallography data have provided CUDC-907 structural evidence of several proposed reaction intermediates and allowed for a more complete understanding of the reaction mechanism of PncA. Apart from the above-mentioned researches, which mainly focus on the catalytic mechanism and the active site residues, the latest crystallographic experiment has highlighted the underestimated importance of protein folding and thermal stability in the MtPncA activity. Indeed, the active-site residues of PncA play a key role in enzymatic catalysis and function, and should be responsible for the activity loss of numerous mutations. But not all the mutations inducing the activity loss occur in the active site, indicative of the significance of the non-active-site residues in PncA function. Among the residues, those in the binding pathway of ligand must be quite crucial for prompting the entrance of ligand to active site and proper orientation. However, a great number of the present researches on PncA pay less attention to the detailed binding process of ligand to PncA, which affirmably hinders the adequate understanding of PncA’s function. The further work of ligand’s binding or unbinding would be necessary for an in-depth illustration of the importance of those non-active-site residues. In this paper, the molecular dynamics simulation methods, which have been successfully applied to many similar researches, were used to explore the binding pathway of NAM to two PncA enzymes from two different bacteria. Based on the steered molecular dynamics simulations, the potential of mean force for three different pulling directions was constructed to determine the most possible binding or unbinding pathway. And then the role of some important residues in the pathway was proposed by calculation and analysis of the interaction energies between the ligand and two enzymes. Some shared structural characteristics of PncA family were presented by the superimposition and comparative analysis among several PncA proteins. Our present work would be helpful for the further research on PncAs and the development of new antituberculosis drugs. It is obvious that the different residues in the possible binding/ unbinding pathways have different effects on the interaction of NAM with two proteins. And the interaction energies between the ligand and all the important residues have been calculated and presented in Figure 5, in which the electrostatic interaction energies are marked as the black curves, the Vdw interaction energies are the green curves, and the total interaction energies are the red ones. The total interaction energies between these residues and NAM all possess the minimum lower than 23 kcal/mol, and display better consistency among six SMD trajectories.