Amino acids are key components of cellular metabolism, serving as the precursors to many metabolites, natural products, and ribosomally and non-ribosomally synthesized peptides and proteins (Figure 1). Therefore, the ability to install useful functional groups onto amino acids in vivo would enable the production of a broad range of new molecules. Our group recently discovered the Fe^II/alpha-ketoglutarate (Fe^II/aKG)-dependent radical halogenase BesD as part of a pathway to produce a terminal alkyne-containing amino acid.¹ BesD modifies the amino acid lysine by installing a halogen on an unactivated sp³ carbon, which is a challenging reaction to achieve selectively using traditional synthetic strategies. Although radical halogenases were discovered close to 15 years ago, only two families have been identified to date.^2,3 Part of the challenge is that radical halogenases seem to have evolved separately within different families and thus share very low sequence homology. In this study, we have solved the crystal structure of BesD to gain insight into its mechanism and used bioinformatics to discover additional members of this novel family of halogenases. The newly discovered halogenases are regioselective, can modify a range of amino acids, and are capable of installing the synthetically useful functional groups -Br and -N₃ in addition to -Cl. Finally, we demonstrated that non-canonical amino acids produced by BesD halogenases can be funneled into enzyme cascades to generate chlorinated heterocycles, diamines, keto-acids, and peptides in vitro.

Figure 1.

Since BesD has a much simpler substrate than the other known families of radical halogenases, it may be a particularly tractable system for studying the mechanism of how enzymes can perform selective halogenation. The Fe^II/aKG-dependent halogenases actually face multiple challenges with regard to selectivity. Beyond the typical problems of substrate selectivity and regioselectivity, halogenases also need to solve the problem of how to achieve reaction pathway selectivity. They use a reactive Fe^IV-oxo species to generate a substrate radical, yielding an intermediate with two possible reaction outcomes (Figure 2). If rebound with the halogen occurs, then the desired halogenated product is produced. However, rebound of the resulting hydroxyl radical can also occur to yield the hydroxylated product. We solved the lysine-bound crystal structure of BesD and performed structure-guided mutagenesis to better understand how the enzyme selectively achieves halogenation instead of hydroxylation. Our results suggest that critical factors for halogenation selectivity include residues that control the precise positioning of lysine in the active site as well as second-sphere residues that may direct the ligand geometry of the Fe-complex.

Figure 2.

We next used BesD as a bioinformatic query to discover new halogenases. We identified homologs of BesD in Pseudomonas and Streptomyces species and examined their genomic contexts. Since many of the neighboring genes encoded for putative amino acid metabolizing enzymes, we reasoned that the newly discovered BesD homologs may also act on amino acids. When we tested the halogenases, we found that they regioselectively chlorinate a range of amino acid substrates. Collectively, the enzymes chlorinate lysine, ornithine, leucine, isoleucine, and norleucine with varied substrate selectivity. Further study of these enzymes may answer questions related to regioselectivity (4- vs 5-halogenation) or mono- vs di-chlorination. 

While the halogenases themselves are interesting, it is also exciting to consider the possibility that these enzymes may be part of fascinating biosynthetic pathways. Indeed, one rationale in Nature for installing a halogen is for it to be lost during downstream chemistry, as is the case for the formation of the cyclopropyl group in coronamic acid (CmaB)⁴, the aromatic ring alkylation in cylindrocyclophane (CylC)⁵, and in alkyne formation (BesD). The identification of several new halogenases in this work expands the scope of radical enzymatic halogenation and sets the stage for the discovery of novel natural products that are formed via radical amino acid halogenases.

You can read more about our work here.

This post was co-authored by Monica E. Neugebauer and Michelle C. Y. Chang.

References

1.        Marchand, J. A. et al. Discovery of a pathway for terminal-alkyne amino acid biosynthesis. Nature 567, 420–424 (2019).

2.        Vaillancourt, F. H., Yin, J., & Walsh, C. T. SyrB2 in syringomycin E biosynthesis is a nonheme Fe^II α-ketoglutarate- and O₂-dependent halogenase. Proc. Natl. Acad. Sci. U. S. A. 102, 10111–10116 (2005).

3.        Hillwig, M. L. & Liu, X. A new family of iron-dependent halogenases acts on freestanding substrates. Nat. Chem. Biol. 10, 921–923 (2014)

4.        Vaillancourt, F. H., Yeh, E., Vosburg, D. A., O’Connor, S. E. & Walsh, C. T. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis. Nature 436, 1191–1194 (2005).

5.        Nakamura, H., Schultz, E. E. & Balskus, E. P. A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis. Nat. Chem. Biol. 13, 916–921 (2017).