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.
