Antibiotics are used since 1940 to fight bacterial infections due to versatility and high efficacy against bacteria. Metallo-β-lactamases (MBLs) are zinc ion dependent enzymes that are responsible for the emergence and spread of β-lactam resistance. Clinically useful MBL inhibitors are not yet available. The increasing number of novel members of this family is threatening to global health care. Sequence identities and structural characteristics at active site has led to the inception of MBL subclasses B1, B2, B3 and recently B4. AIM-1 (Adelaide IMipenemase-1) is a novel and poorly characterized MBL that was found in Adelaide, Australia and found to be similar in many ways to numerous other enzymes that pose a threat to human health because of their ability to protect bacteria from penicillin like compounds. Being an exclusive member of B3 subclass AIM-1 is suggested as a potential candidate for broad spectrum MBL inhibitor. In present study, a series of mutant forms of AIM-1 was produced by site-specific mutagenesis. D155, D220 and T223 were selected as second shell metal coordinated residues in case of Zn1 whereas D120 was selected as primary ligand. The native and mutant enzymes were then expressed and purified so that the kinetic properties of the mutant enzymes could be compared with those of the native enzyme. Metal chelators were used to remove the metals from the native and mutant forms of the enzyme so that ITC (isothermal titration calorimetry) could be used to determine the metal binding constants. Kinetic data suggest that Asp120 and second shell ligands have showed clear effects and are important for enzymatic activity. In case of Zn1 D to N mutagenesis in second metal coordinates accommodate the smaller substrate molecules like Carbapenems and can effect metal binding whereas D to A mutation severely impaired catalytic rate. Two exothermic metal binding events were observed where one is stronger and other is weaker. Later, two putative MBLs were identified by protein database search, in silico characterized and compared with AIM-1. Information gained from this study may also be useful in the design of inhibitors that could be used as anti-bacterial agents.