Two Flavoenzymes Catalyze the Post-Translational Generation of 5-Chlorotryptophan and 2-Aminovinyl-Cysteine during NAI-107 Biosynthesis

Abstract

Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides containing thioether rings. In addition to these crosslinks, the clinical candidate lantibiotic NAI-107 also possesses a C-terminal S-[(Z)-2-aminovinyl]-D-cysteine (AviCys) and a unique 5-chloro-L-tryptophan (ClTrp) moiety linked to its potent bioactivity. Bioinformatic and genetic analyses on the NAI-107 biosynthetic gene cluster identified mibH and mibD encoding flavoenzymes responsible for the formation of ClTrp and AviCys, respectively. The biochemical basis for the installation of these modifications on NAI-107 and the substrate specificity of either enzyme is currently unknown. Using a combination of mass spectrometry, liquid chromatography, and bioinformatic analyses we demonstrate that MibD is an FAD-dependent Cys decarboxylase and that MibH is an FADH2-dependent Trp halogenase. Most FADH2-dependent Trp halogenases halogenate free Trp, but MibH was only active when Trp was embedded within its cognate peptide substrate deschloro NAI-107. Structural comparison of the 1.85-Å resolution crystal structure of MibH with other flavin-dependent Trp halogenases revealed that subtle amino acid differences within the MibH substrate binding site generates a solvent exposed crevice presumably involved in determining its unusual substrate specificity.

Chlorinated Natural Products and Related Halogenases

“…The gene mibH was later identified as an FDH reacting in the early stage of NAI‐107 biosynthesis. Different from other reported tryptophan halogenases, MibH cannot work on free tryptophan . Tests on the other lanthipeptides analogues showed negative results, indicating the strict substrate specificity of MibH.…”

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

“…4A, 7 ) without the need for a ACP (68). The FDH MibH was recently discovered and found to chlorinate a lanthipeptide (~1kD) (74). Several FDHs, including Rdc2/RadH (59), CazI (61), and Bmp5 (58), have been found to halogenate free phenol-containing compounds (63) (Fig.…”

“…When the structures of CmlS (71), CndH (77) (an FDH thought to halogenate a phenolic substrate), PltA (78), MibH (74), and RebH (40) are compared, many structurally conserved regions are also found in the oxygenase PHBH (71). Structural analysis displays high conservation of the FAD-binding domain and halogenation active site; however, the C-terminus varies between CmlS, CndH, PltA, MibH, and Trp-FDHs (Fig.…”

“…On the other hand, the crystal structure of MibH, which halogenates a lantipeptide (~1 kDa), does indeed have a much larger substrate binding pocket than other FDHs (718.5 Å 2 larger), even in the absence of bound substrate (74). Although it appears that MibH, like Trp-FDHs, does not need to undergo large conformational changes to halogenate this peptide, crystallization of substrate bound structure will be necessary to confirm this.…”

Bromotryptophans and their incorporation in cyclic and bicyclic privileged peptides

“…Of note, a unique 5‐ClTrp moiety, inserted through biocatalysis, is present in a clinical candidate with antimicrobial bioactivity. Here, the chlorine substituent is responsible for its increased …”