Biochemical Studies of a Cyanobacterial Halogenase Support the Involvement of a Dimetal Cofactor

Abstract

Halogenation is a prominent transformation in natural product biosynthesis, with over 5000 halogenated natural products known to date. Biosynthetic pathways accomplish the synthetic challenge of selective halogenation, especially at unactivated sp³ carbon centers, using halogenase enzymes. The halogenase CylC, discovered as part of the cylindrocyclophane (cyl) biosynthetic pathway, performs a highly selective chlorination reaction on an unactivated sp³ carbon center and is proposed to use a dimetal cofactor. Putative dimetal halogenases are widely distributed across cyanobacterial biosynthetic pathways. However, rigorous in vitro biochemical and structural characterization of these enzymes has been challenging. Here, we report additional bioinformatic analyses of putative dimetal halogenases and the biochemical characterization of a newly identified CylC homologue. Site-directed mutagenesis identifies highly conserved putative metal-binding residues, and Mössbauer spectroscopy provides direct evidence for the presence of a diiron cofactor in these halogenases. These insights suggest mechanistic parallels between diiron and mononuclear nonheme iron halogenases, with the potential to guide further characterization and engineering of this unique subfamily of metalloenzymes.

1

Radical halogenases functionalize unactivated carbon centers. (A) Overall reaction and examples of radical halogenation by mononuclear nonheme iron halogenases. (B) Putative diiron halogenases biosynthesize fatty acid-derived cyanobacterial natural products. (C) Characterization of active site residues and spectroscopy suggest putative diiron halogenases use a diiron cofactor for radical halogenation.

2

Bioinformatic analyses of CylC homologues predict amino acids involved in dimetal cofactor binding and identify a potential chloride binding site. (A) Protein sequence alignment of AurF with select CylC homologues reveals a set of putative active site residues. (B) AlphaFold3 predicted structure of CylC contains the FDO-like fold found in AurF and a dimetallocofactor with an open coordination site for halide binding. Italicized residues represent active site residues that are predicted to not directly participate in metal binding.

3

NocO chlorinates the terminal position of NocM-tethered dodecanoyl thioester. (A) AlphaFold3 predicted structure of the NocM/NocO complex. (B) In vitro assay for halogenation of 1 by NocO (Fdx = ferredoxin, Fdr = ferredoxin reductase). (C) Representative extracted ion chromatogram (EIC) of trypsin digested product 3 in full in vitro reactions and reactions lacking the electron source NADPH. (D) Representative MS spectra of 3 extracted from EICs in (C). (E) MS/MS fragmentation of product in reactions supplemented with NocL-loaded NocM-12,12,12-d ₃-dodecanoyl thioester shows NocO selectively halogenates the terminal carbon of the acyl chain of 1. Assays and negative controls were performed in triplicate for experiments in (C) and (D), assays and negative controls were performed in duplicate for the isotopic labeling experiment in (E).

4

Mutation of predicted metal binding residues and Mössbauer spectroscopy support the presence of a diiron cofactor in NocO. (A) Putative active site residues in NocO identified in an AlphaFold3-predicted structure. Residues shown in white are predicted to be not directly involved in metal binding. (B) Representative EICs for 3 show that active site variants are catalytically inactive. (C) Certain active site variants show decreased iron loading relative to WT NocO. Assays and corresponding negative controls were performed in triplicate, bars represent mean ± SD. (D) 4.2-K/53-mT Mössbauer spectrum of ⁵⁷Fe-enriched, aerobically isolated NocO. The experimental spectrum is depicted in black vertical bars of heights reflecting the standard deviations of the absorption values during spectral acquisition. The blue, red, and yellow lines are quadrupole doublet simulations illustrating the fractional contributions from the different diiron­(III)-species quoted in the text. The black arrows represent the high-energy lines of the Fe­(II)-species, as quoted in the text. The parameters of the different iron-species used for simulation are summarized in Table S7.