Breaking a Strong Amide Bond: Structure and Properties of Dimethylformamidase.

Abstract

Dimethylformamidase (DMFase) breaks down the human-made synthetic solvent N,N-dimethyl formamide(DMF) used extensively in industry(1). DMF is not known to exist in nature and was first synthesized in 1893. In spite of the recent origin of DMF, certain bacterial species such as Paracoccus, Pseudomonas, and Alcaligenes have evolved pathways to breakdown DMF and use them as carbon and nitrogen source for growth(2, 3). The work presented here provides a molecular basis for the ability of DMFase from Paracoccus to function in exacting conditions of high solvent concentrations, temperature and ionic strength to catalyze the hydrolysis of a stable amide bond. The structure reveals a multimeric complex of the 2{beta}2 type or (2{beta}2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed folds and as yet of unknown evolutionary origin. The active site is made of a distinctive mononuclear iron that is coordinated by two tyrosine residues and a glutamic acid residue. The hydrolytic cleavage of the amide bond is catalyzed at the Fe3+ site with a proximal glutamate probably acting as the base. The change in the quaternary structure is salt dependent with high salt resulting in the larger oligomeric state. Kinetic characterization reveals an enzyme that shows cooperativity between subunits and the structure provides clues on the interconnection between the active sites. Significance StatementN,N-dimethyl formamide(DMF) is a commonly used industrial solvent that was first synthesized in 1893. The properties that make DMF a highly desired solvent also makes it a difficult compound to breakdown. Yet, certain bacteria have evolved to survive in environments polluted by DMF and have enzymes that breakdown DMF and use it as their carbon and nitrogen source. The molecular structure of the enzyme that breaks down the stable amide bond in these bacteria, reveals two new protein folds and a unique mononuclear iron active site. The work reported here provides the structural and biochemical framework to query the evolutionary origins of the protein, as well as in engineering this enzyme for use in bioremediation of a human made toxic solvent.