Abstract
AbstractN,N‐dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2β2 or (α2β2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side‐chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.
Highlights
Dimethylformamide (DMF) is an organic solvent commonly used in chemical synthesis, leather, printing, and petrochemical industries
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
A large cavity is observed at the interface between the large and small subunits, which leads towards the active site (Fig. 4B)
Summary
Dimethylformamide (DMF) is an organic solvent commonly used in chemical synthesis, leather, printing, and petrochemical industries. Its polar nature accords properties similar to water, and its physicochemical features make it versatile(miscible in many organic solvents and water, a high boiling point of 153 °C, etc.) [1, 6,7,8]. It is a known hepatotoxic and ecotoxic agent[9, 10]. We performed site-directed mutagenesis of specific residues to test their role in metal binding and catalysis Together, these data allow us to propose a plausible mechanism and a molecular explanation for the narrow substrate specificity of the enzyme
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