Genome mining for the discovery of peptide halogenases and their biochemical characterization

Abstract

While halogenation is one of the most versatile C-H functionalization strategy, regiospecific halogenation of peptides and proteins is outside the purview of traditional chemical catalysis. Enzymes that participate in the biosynthesis of ribosomally synthesized and post-translationally modified peptides and proteins can bridge this gap and offer a biocatalytic route for residue-specific incorporation of halogen handles onto amino acid side chains. Protocols described herein provide a guided approach for the discovery of peptide halogenases in the context of natural product biosynthetic gene clusters, and the preliminary reconstitution of their activity using a bacterial heterologous host. Also described are mass spectrometry-based analytical procedures and data analysis workflows that allow for deconvolution of halide specificity and preliminary insights into peptidic natural product biosynthetic schemes. As the available genomic data expands at a rapid rate, the methodology described here will enable the discovery and characterization of new halogenases that can be valuable partners in chemoenzymatic diversification of peptides and proteins.

Keywords: Genome mining; Halogenase; Mass spectrometry; RiPPs.

Figure 1:

Halogenated peptidic natural products vancomycin and bromodarobactin with favorable antimicrobial properties.

Figure 2:

Activity of the tryptophan-6-brominase SrpI. The cyclodehydratase SrpC dehydratases the three contiguous Cys residues in the SrpE pentapeptide core to install azoline heterocycles followed by bromination by SrpI. Note that SrpI was found to be a physiological brominase such that it did not characterize chlorination (Nguyen et al. 2021).

Figure 3:

EFI-generated SSN illustrating similarity-based clustering of protein sequences. The node corresponding to the input SrpI sequence is illustrated in yellow.

Figure 4:

The GND visualized using the online EFI toolkit corresponding to the SrpI cluster 2. The central gene colored red is the SrpI-like halogenase encoding gene. Hovering over the genes demonstrates their functional annotations. The BGCs illustrated here are derived from a marine sponge-associated Proteobacteria, as has been reported for srp BGCs previously.

Figure 5:

The GND visualized using the online EFI toolkit corresponding to the SrpI cluster 5. Hovering over one of the genes in the neighborhood of the SrpI-like halogenase encoding gene shows the annotation as a lanthionine synthetase.

Figure 6:

The mpp BGC. The genes mppM, mppE, and mppI encode ATP-dependent lanthionine synthetase, the RiPP precursor peptide, and a flavin-dependent halogenase, respectively. The putative functional annotations of the mpp open reading frames are denoted. The genes colored cyan encode putative membrane transporters.

Figure 7:

Scheme for modification of the MppE precursor peptide by lanthionine synthetase MppM and flavin-dependent halogenase MppI. It is not known, a priori, whether MppI is a chlorinase or a brominase.

Figure 8:

The isotopic distribution of [M+2H]²⁺ ions corresponding to the LahT150-digested MppE peptides: (A) unmodified MppE core, (B) MppM-modified macrocyclic MppE core, (C) macrocyclized and monobrominated MppE core, (D) linear and monobrominated MppE core, (E) macrocyclized and dibrominated MppE core, and (F) linear and dibrominated MppE core. These products were observed when mppE was coexpressed with mppM and mppI in E. coli. Note that no chlorinated products were detected.

Figure 9:

EICs for [M+2H]²⁺ ions demonstrating the presence of MppE-derived RiPPs when mppE was coexpressed with mppM and mppI in E coli. From bottom to top, the following chromatograms are shown: 1–linear unmodified MppE core, 2–MppM-modified macrocyclic MppE core, 3–linear and monobrominated MppE core, 4–linear and dibrominated MppE core, 5–macrocyclized and monobrominated MppE core, and 6–macrocyclized and dibrominated MppE core. It is evident that the monobrominated products predominate, and that while this experiment establishes the bromination activity of MppI, it does not inform whether the physiological substrate for MppI is the linear unmodified MppE peptide, or MppM-modified macrocyclic MppE peptide.

Figure 10:

Proposed routes for oxidative halogenation by flavin-dependent halogenases. The oxidized flavin cofactor (Fl_ox) is reduced, and the reduced flavin cofactor (Fl_red) coordinates with molecular oxygen to generate the flavin-C4a peroxide (Fl_C4aOO−). The addition of halide displaces one of the oxygen atoms, leading to the formation of a Fl_C4aOX intermediate which is resolved either by haloamine intermediate formation (red arrows) or by displacement of hypohalous acid in which the Lys side chain acts as the catalytic base to protonate the hypohalite. In either scenario, the Lys side chain delivers the halonium to the hydrocarbon binding site that is distal to the flavin cofactor.