Structural basis for halogenation by iron- and 2-oxo-glutarate-dependent enzyme WelO5

Abstract

A 2.4-Å-resolution x-ray crystal structure of the carrier-protein independent halogenase, WelO5, in complex with its welwitindolinone precursor substrate, 12-epi-fischerindole U, reveals that the C13 chlorination target is proximal to the anticipated site of the oxo group in a presumptive cis-halo-oxo-iron(IV) (haloferryl) intermediate. Prior study of related halogenases forecasts substrate hydroxylation in this active-site configuration, but x-ray crystallographic verification of C13 halogenation in single crystals mandates that ligand dynamics must reposition the oxygen ligand to enable the observed outcome. Ser189Ala WelO5 effects a mixture of halogenation and hydroxylation products, showing that an outer sphere hydrogen bonding group orchestrates ligand movements to achieve a configuration that promotes halogen transfer.

Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase

“…A recent structure of an Fe/2OG chlorinase, WelO5, which harbors a Phe at the site occupied by Arg 334 in VioC, suggests use of a geometrically distinct ferryl located in the equatorial plane to achieve selective halogenation in this system. 19 For this reason, the coordination geometry observed in the VioC vanadium complex, in which a site cis to the present location of the oxo is accessible for its possible migration during or after ferryl formation and controlled by interaction of the ligand (or lack thereof) with outer-sphere residues, is intriguing. Notably, the amino acid hydroxylase, SadA, which has Phe at the position equivalent to Arg 334 in VioC, can be modified to halogenate its substrate, 39 demonstrating successful reprogramming of function via site-directed mutagenesis in an Fe/2OG enzyme.…”

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

“…The only structural information available regarding the interaction between enzyme and a CP-tethered substrate is an NMR structure of a CP domain from the curacin biosynthetic pathway and mapping of the CP residues that modulate Cur Hal activity based on mutational analyses. 276 However, the discovery of WelO5 has facilitated the attainment of a co-crystal structure with the substrate 12- epi -fischerindole U, 271 and furthered the elucidation of the mechanism of halogenation/hydroxylation control, as discussed below.…”

Development of Halogenase Enzymes for Use in Synthesis

“…This class were initially believed to be of little value for biocatalysis due their selectivity for carrier-protein tethered amino acid substrates, although the recent discovery of a α-Fe(II)/α-KG dependent halogenase capable of catalysing the regio-and stereo-selective halogenation of freely-diffusing substrates offers fresh promise for this class of halogenase. [108][109][110][111] The substrate scope of this class is yet to be explored extensively, as is their amenability to engineering for modulation of substrate scope and selectivity, but the limited number of examples thus far is promising and is likely to broaden should further enzymes of this class be discovered. The demonstration that mutagenesis of an Fe(II)/α-KG dependent hydroxylase can be engineered to provide a halogenase biocatalyst is also promising because this could allow the engineering of other α-KG/Fe(II)-dependent hydroxylases, which accept a broad range of freely-diffusing substrates, into additional biocatalysts for the halogenation of aliphatic C-H bonds.…”

“…The recent publication of the WelO5 crystal structure revealed the close proximity of the C13 halogenation site to the putative cis-halo-oxo-Fe IV complex which confirms its preferred activity towards halogenation ( Figure 10C). 111 Unlike the standard α-KG hydroxylases, WelO5 contains a glycine (Gly166) at the sequence position of an aspartate or glutamate ligand. Accordingly, it was shown that the G166D variant exclusively gives C13 hydroxylation, as predicted.…”