Towards a Mechanism for the Hydrolysis of Dimethylformamide by the Enzyme Dimethylformamidase

Abstract

N,N-dimethylformamide (DMF) is a widely used organic solvent due to its hydrophilic and highly stable nature. However, it is also a potent pollutant which requires bioremediation solutions. The enzyme Dimethylformamidase (DMFase), extracted from the bacterium Paracoccus sp. strain DMF, can cleave the highly stable amide bond present in DMF. The structure of DMFase was recently published, showing that it is an α2β2 heterotertameric complex with an unusual mononuclear Fe3+ center. A reaction mechanism has been proposed, however, the exact role of the residues present in the active site is not yet known due to the unavailability of the structure of an enzyme-substrate complex. In this project, I studied the role of different residues in the active site using site-directed mutagenesis, isothermal calorimetry and structural analysis. The results from the activity assays of the mutants along with the structure of the E657D mutant provide further support to the proposed mechanism. Mutagenesis studies have also revealed a few other residues that can play a role in the catalytic reaction.