The enantiomers of 2-azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) are key chiral synthons in the synthesis of antiviral drugs such as carbovir and abacavir. (+)-γ-Lactamase can be used as a catalyst in the enzymatic preparation of optically pure (−)-γ-lactam. Here, a (+)-γ-lactamase discovered from Bradyrhizobium japonicum USDA 6 by sequence-structure guided genome mining was cloned, purified and characterized. The enzyme possesses a significant catalytic activity towards γ-lactam. The active site of the (+)-γ-lactamase was studied by homologous modeling and molecular docking, and the accuracy of the prediction was confirmed by site-specific mutagenesis. The (+)-γ-lactamase reveals the great practical potential as an enzymatic method for the efficient production of carbocyclic nucleosides of pharmaceutical interest.