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 -ketoglutarate dependent oxygenases (FeDOs) catalyze 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 a FeDO facial triad variant, SadX, and the effects of that binding on HO• vs. X• rebound. We establish that chloride and bromide not only enable non-native halogenation reactions but 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 led 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 FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving those ligands, and inherent rebound preferences relative to HO• that will aid efforts to develop non-native catalysis using these enzymes.

Directed Evolution of a Fe(II)- and α-Ketoglutarate-Dependent Dioxygenase for Site-Selective Azidation of Unactivated Aliphatic C-H Bonds

“…Analysis of product distributions for SadX variants in the azidase lineage revealed subtle relationships between the site and chemoselectivity of the azidation, chlorination, and hydroxylation reactions catalyzed by these enzymes (Figure 2C). As previously reported, [47] 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 βposition of the substrate, while azidation occurs at the 𝛾-position.…”

“…Recently, we reported that expression of this enzyme is improved by fusing it to maltose binding protein (MBP) to generate MBP-SadA D157G (hereafter SadX), and we evaluated SadX activity on N-succinyl leucine (1a) in the presence of different anions. [47] SadX exhibits relatively broad scope with respect to X • rebound (X • = Cl • , Br • , N3 • , NCO • ), suggesting that it could serve as a useful platform for non-native FeDO catalysis. The improved expression of SadX allowed for LC/MS analysis of non-native X • rebound in crude E. coli cell lysate containing the desired Xsalt and excess ascorbate [48] (Figure 2A).…”

“…We then established that even SadX gives comparable yield and selectivity toward azidation and chlorination. [47] Saturation mutagenesis of active site residues in SadX that were reported to improve SadA-catalyzed oxygenation of Nsuccinyl-3,4-dimethoxyphenylalanine (G79 and F261) was therefore pursued, but improved azidation yields were not observed. [45] Given that 1-VH provided similar yield and selectivity for chlorination and azidation, we opted to continue evolving the latter reaction (Figure 2B).…”

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

“…In this study, we explored the binding of different anions, X – , to an FeDO facial triad variant, SadX, and the effects of anion binding on HO • and X • rebound. Spectrophotometric titrations revealed that several anions known to bind the Fe­(II) center of other FeDHs and FeDO facial triad variants also bound to SadX, and evidence for fluoride binding not reported for other systems was also observed.…”

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase

“…Their ability to chlorinate and brominate a variety of substrates including amino acids, nucleotides, and natural products make them a highly versatile biocatalytic platform that invite further optimization. In addition to halogenation, these enzymes have also been shown to functionalize C-H bonds with azide (Büchler et al, 2022;Chan et al, 2022;Matthews et al, 2014;Neugebauer et al, 2019) and nitrite (Matthews et al, 2014), albeit with disappointingly low yields. A variety of methods have been used to determine substrate affinity in these and related classes of enzymes, including tryptophan fluorescence (Martin et al, 2019), SPR (Hu et al, 2015), and UV-Vis spectral shift-based methods (Chan et al, 2022;Matthews et al, 2014;Price et al, 2003;Ryle et al, 1999) to name a few.…”