A 2‐Tyr‐1‐carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase

Abstract

N,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.

Equilibrium dynamics of a biomolecular complex analyzed at single-amino acid resolution by cryo-electron microscopy

“…To discard the possibility that the MVM capsid might be an unusual complex for which those limiting factors had no significant effect on dynamics-related parameters, we tested the same approach for other viral and cellular protein complexes from the PDB (Berman et al, 2000) and EMDB (Lawson et al, 2016) repositories. Four different assemblies solved to atomic or near atomic resolution by both X-ray crystallography and cryo-EM were selected: β-galactosidase (Bartesaghi et al, 2018; Juers et al, 2000), dimethylformamidase [DMFase, (Arya et al, 2020; Vinothkumar, Arya, Ramanathan, & Subramanian, 2021)], apoferritin (Masuda, Goto, Yoshihara, & Mikami, 2010; Yip, Fischer, Paknia, Chari, & Stark, 2020) and porcine circovirus-2 [PCV-2, (Liu, Guo, Wang, Li, & Jiang, 2016; Mo et al, 2019)]. For every macromolecular complex a clear correlation between LR values and relative B-factors was found (Fig.…”

Expression, purification and crystallization of N-acetyl-(R)-β-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 and structure determination of the Burkholderia enzyme

“…KUFA-1 and Paracoccus sp. strain DMF have the same length as each other and 99% identical residues, and require not only the large subunit but also another small subunit for their activity (Hasegawa et al, 1999;Arya et al, 2020). From structural studies using cryo-EM and crystallography, it was revealed that the DMFase derived from Paracoccus sp.…”

“…From structural studies using cryo-EM and crystallography, it was revealed that the DMFase derived from Paracoccus sp. strain DMF (parDMFase) forms an () 2 heterotetramer composed of two large 84 kDa subunits and two small 16 kDa subunits (PDB entry 6lvv; Arya et al, 2020). Although the function of the small subunit remains unclear, it is most likely to play a role in structural stabilization of an enzymatically active form (Arya et al, 2020).…”

“…strain DMF (parDMFase) forms an () 2 heterotetramer composed of two large 84 kDa subunits and two small 16 kDa subunits (PDB entry 6lvv; Arya et al, 2020). Although the function of the small subunit remains unclear, it is most likely to play a role in structural stabilization of an enzymatically active form (Arya et al, 2020). On the other hand, B--FAA expressed in E. coli exhibited activity without requiring another subunit, showing that it has a different quaternary structure to the DMFases.…”

Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3

“…Cytochrome P450 enzymes (P450s) are a superfamily of heme proteins involved in drug metabolism, xenobiotic detoxification, and steroid biosynthesis [ 1 , 2 ]. P450s are promising biocatalysts for organic synthesis, drug discovery, and bioremediation because of their versatile catalytic oxyfunctionalizations of a variety of substrates [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. However, one of the limitations of applying P450s as in vitro biocatalysts is that most P450s require reduced nicotinamide cofactors NAD(P)H and a complex system of electron transport partners (redox partners) to activate molecular oxygen for the formation of active species.…”