TY - JOUR
T1 - Biosynthesis of Macrocyclic Peptides with C‑Terminal β‑Amino-α-keto Acid Groups by Three Different Metalloenzymes
AU - Nguyen, Dinh T.
AU - Zhu, Lingyang
AU - Gray, Danielle L.
AU - Woods, Toby J.
AU - Padhi, Chandrashekhar
AU - Flatt, Kristen M.
AU - Mitchell, Douglas A.
AU - van der Donk, Wilfred A.
N1 - Publisher Copyright:
© 2024 The Authors. Published by American Chemical Society.
PY - 2024/5/22
Y1 - 2024/5/22
N2 - Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical Sadenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C−C cross-link between two Tyr residues with the B12-rSAM generating β-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the β-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.
AB - Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical Sadenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C−C cross-link between two Tyr residues with the B12-rSAM generating β-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the β-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.
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U2 - 10.1021/acscentsci.4c00088
DO - 10.1021/acscentsci.4c00088
M3 - Article
C2 - 38799663
AN - SCOPUS:85191378897
SN - 2374-7943
VL - 10
SP - 1022
EP - 1032
JO - ACS Central Science
JF - ACS Central Science
IS - 5
ER -