Structure of apo flavin-dependent halogenase Xcc4156 hints at a reason for cofactor-soaking difficulties.

Christiane Widmann,Hartmut H Niemann,Mohamed Ismail,Norbert Sewald

doi:10.1107/s2059798320007731

Christiane Widmann, Hartmut H Niemann + Show 2 more

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https://doi.org/10.1107/s2059798320007731

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Abstract

Flavin-dependent halogenases regioselectively introduce halide substituents into electron-rich substrates under mild reaction conditions. For the enzyme Xcc4156 from Xanthomonas campestris, the structure of a complex with the cofactor flavin adenine dinucleotide (FAD) and a bromide ion would be of particular interest as this enzyme exclusively brominates model substrates invitro. Apo Xcc4156 crystals diffracted to 1.6 Å resolution. The structure revealed an open substrate-binding site lacking the loop regions that close off the active site and contribute to substrate binding in tryptophan halogenases. Therefore, Xcc4156 might accept larger substrates, possibly even peptides. Soaking of apo Xcc4156 crystals with FAD led to crumbling of the intergrown crystals. Around half of the crystals soaked with FAD did not diffract, while in the others there was no electron density for FAD. The FAD-binding loop, which changes its conformation between the apo and the FAD-bound form in related enzymes, is involved in a crystal contact in the apo Xcc4156 crystals. The conformational change that is predicted to occur upon FAD binding would disrupt this crystal contact, providing a likely explanation for the destruction of the apo crystals in the presence of FAD. Soaking with only bromide did not result in bromide bound to the catalytic halide-binding site. Simultaneous soaking with FAD and bromide damaged the crystals more severely than soaking with only FAD. Together, these latter two observations suggest that FAD and bromide bind to Xcc4156 with positive cooperativity. Thus, apo Xcc4156 crystals provide functional insight into FAD and bromide binding, even though neither the cofactor nor the halide is visible in the structure.

Highlights

The activation of C—H bonds in electron-rich organic compounds is an important step that enables their use in a range of downstream applications such as metal-catalyzed cross-coupling reactions or modifications via nucleophilic substitution (Runguphan & O’Connor, 2013; Frese et al, 2016; Durak et al, 2016)
We recently identified four halogenases that act on indole derivatives but not on free tryptophan
The xcc4156 gene was amplified from a pET-28a plasmid (Ismail et al, 2019) and cloned into a pETM-11 expression vector which contains a hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site

Summary

Introduction

The activation of C—H bonds in electron-rich organic compounds is an important step that enables their use in a range of downstream applications such as metal-catalyzed cross-coupling reactions or modifications via nucleophilic substitution (Runguphan & O’Connor, 2013; Frese et al, 2016; Durak et al, 2016). Flavin-dependent halogenases (FDHs) have come into focus as a clean alternative because they halogenate their substrates under much milder conditions using halide salts and molecular oxygen at 25 C and pH 7 (Keller et al, 2000; Yeh et al, 2005). Their reaction mechanism has been investigated both in vitro (Dong et al, 2005; Yeh et al, 2006, 2007; Flecks et al, 2008; Fraley et al, 2017) and in silico

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