Maleamate amidohydrolase (NicF) is a key enzyme in vitamin B3 metabolism that catalyzes the hydrolysis of maleamate to produce maleic acid and ammonia. Unlike most members from the amidohydrolase superfamily it does not require a metal ion. Here, we use multiscale computational enzymology to investigate the catalytic mechanism, substrate binding, oxyanion hole, and roles of key active site residues of NicF from Bordetella bronchiseptica. In particular, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) and QTAIM methods have been applied. The mechanism of the NicF-catalyzed reaction proceeds by a nucleophilic addition-elimination sequence involving the formation of a thioester enzyme intermediate (IC2 in stage 1) followed by hydrolysis of the thioester bond to form the products (stage 2). Consequently, the formation of IC2 in stage 1 is the rate-limiting step with a barrier of 88.8 kJ·mol-1 relative to the reactant complex, RC. Comparisons with related metal-dependent enzymes, particularly the zinc-dependent nicotinamidase from Streptococcus pneumonia (SpNic), have also been made to further illustrate unique features of the present mechanism. Along with -NH- donor groups of the oxyanion hole (i.e., HN-Thr146, HN-Cys150), the active site β-hydroxyl of threonine (HO-βThr146) is concluded to play a role in stabilizing the carbonyl oxygen of maleamate during the mechanism.