Non-Native Anionic Ligand Binding and Reactivity in Engineered Variants of the Fe(II)- and α-Ketoglutarate-Dependent Oxygenase, SadA

Abstract

Mononuclear non-heme Fe­(II)- and α-ketoglutarate-dependent oxygenases (FeDOs) catalyze a site-selective C–H hydroxylation. Variants of these enzymes in which a conserved Asp/Glu residue in the Fe­(II)-binding facial triad is replaced by Ala/Gly can, in some cases, bind various anionic ligands and catalyze non-native chlorination and bromination reactions. In this study, we explore the binding of different anions to an FeDO facial triad variant, SadX, and the effects of that binding on HO• vs X• rebound. We establish not only that chloride and bromide enable non-native halogenation reactions but also that all anions investigated, including azide, cyanate, formate, and fluoride, significantly accelerate and influence the site selectivity of SadX hydroxylation catalysis. Azide and cyanate also lead to the formation of products resulting from N3 •, NCO•, and OCN• rebound. While fluoride rebound is not observed, the rate acceleration provided by this ligand leads us to calculate barriers for HO• and F• rebound from a putative Fe­(III)­(OH)­(F) intermediate. These calculations suggest that the lack of fluorination is due to the relative barriers of the HO• and F• rebound transition states rather than an inaccessible barrier for F• rebound. Together, these results improve our understanding of the FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving these ligands, and inherent rebound preferences relative to HO• that will aid efforts to develop non-native catalysis using these enzymes.

Directed Evolution of an Iron(II)‐ and α‐Ketoglutarate‐Dependent Dioxygenase for Site‐Selective Azidation of Unactivated Aliphatic C−H Bonds**

“…Nonetheless, this analysis revealed subtle relationships between the site and chemoselectivity of the azidation, chlorination, and hydroxylation reactions catalyzed by these enzymes (Figure 2C). As previously reported, [49] SadX prefers hydroxylation to chlorination by over two-fold, and a similar preference for hydroxylation over azidation is observed. Notably, however, hydroxylation and chlorination occur at the β-position of the substrate, while azidation occurs at the γ-position.…”

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

“…Without such bias, the only demonstration of a selective, catalyst-controlled derivatization is the seminal report by Davies and co-workers − on rhodium-catalyzed asymmetric carbene insertion into hydrocarbon C–H bonds (also see ref ). Recently, selective azidation and nitration of unactivated C–H bonds have been demonstrated with non-heme iron enzymes. − Here, we establish that P450 enzymes can be engineered for the introduction of nitrogen at unbiased, unactivated C­(sp 3 )–H centers with high site and stereoselectivity, revealing a new biochemical pathway for C–H functionalization that, analogous to P450-catalyzed C–H hydroxylation, can be tuned and diversified by evolution (Figure ).…”

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

“…Recently, selective azidation and nitration of unactivated C-H bonds has been demonstrated with non-heme iron enzymes. [33][34][35] Here, we establish that P450 enzymes can be engineered for introduction of nitrogen at unbiased, unactivated C(sp 3 )-H centers with high site and stereoselectivity, revealing a new biochemical pathway for C-H functionalization that, analogous to P450-catalyzed C-H hydroxylation, can be tuned and diversified by evolution (Figure 1).…”