Cytochromes P450 Associated with the Biosyntheses of Ribosomally Synthesized and Post-translationally Modified Peptides

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of exponentially increased natural products with characteristic chemical structures, topologies, and biosynthetic mechanisms as well as exceptional bioactivities including antibacteria, antitumors, and antiviruses. The biosynthesis of RiPP proceeds via a ribosomally assembled precursor peptide that undergoes varied post-translational modifications to generate a mature peptide. Cytochrome P450 (CYP or P450) monooxygenases are a superfamily of heme-containing enzymes that span a wide range of secondary metabolite biosynthetic pathways due to their broad substrate scopes and excellent catalytic versatility. In contrast to the enormous quantities of RiPPs and P450s, the P450 associated RiPP biosynthesis is comparatively limited, with most of their functions and timings remaining mysterious. Herein, this Review aims to provide an overview on the striking roles of P450s in RiPP biosyntheses uncovered to date and to illustrate their remarkable functions, mechanisms, as well as remaining challenges. This will shed light on novel P450 discovery and characterizations in RiPP biosyntheses.

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

“…We sought out an RRE-dependent RiPP BGC encoding multiple uncharacterized metalloenzymes, given their unparalleled ability to catalyze difficult and diverse chemical transformations. − A BGC obtained from RRE-Finder that caught our interest was from B. thailandensis E264 and is composed of a sequence-unique substrate peptide (ApyA, NCBI accession code: ABC34935.1), an MNIO enzyme (ApyH, ABC35712.1), an RRE-containing protein (ApyI, ABC34269.1), a methyltransferase (ApyS, ABC34725.1), an RRE-containing cobalamin- and radical S -adenosyl- l -methionine-dependent enzyme (B12-rSAM, ApyD, ABC34219.1), , and a cytochrome P450 enzyme (ApyO, ABC35200.1) (Figure and supplementary data set 1). , To obtain homologous BGCs, we performed a BLASTP search of each enzyme against the nonredundant NCBI protein database. The genome neighborhood and putative precursor peptides corresponding to the collected homologues were obtained using RODEO, and BGCs encoding putative precursor(s) were analyzed.…”

“…To the best of our knowledge, ApyD is the first enzyme reported to methylate the Cβ of Tyr. In contrast, ApyO catalyzes biaryl cross-linking between two Tyr residues, a reaction that has been observed with the non-RiPP natural products arylomycin (AryC) and mycocyclosin (CYP121), albeit with different substrates and stereochemical outcomes. , C–C bond formations involving two aromatic rings have been reported to be catalyzed by cytochrome P450 enzymes in RiPP biosynthesis, ,,, with a recent example (SlyP) catalyzing a similar cross-link as ApyO but without information on atropisomerism . Some of the precursor peptides in the identified BGCs here have other aromatic residues in the second and fourth positions from the C-terminus (Trp/Tyr; see supplementary data set 1), suggesting that other biaryl linkages will be formed.…”

Genome Mining for New Enzyme Chemistry

“…Besides their natural catalytic activity, directed evolution techniques have been extensively employed to engineer P450 enzymes to catalyze unnatural transformations . In RiPP biosynthesis, P450-catalyzed reactions are frequently observed, with most identified retrospectively after traditional natural product isolation efforts. , We focus here on noncanonical cytochrome P450-catalyzed transformations. Examples include macrocyclization during the biosynthesis of tryptorubin, , cittilins, biarylitides, and the recently discovered cihunamides, which display a C–N bond between C7 in Trp (residue i) and the N1 of another Trp (i+3).…”

Bacterial cyclophane-containing RiPPs from radical SAM enzymes

“…Biaryl RiPPs that include cittilins, 17 biarylitides, 18 myxarylins, 19 and bitryptides 20 are formed by P450 oxygenases, and are not included in this highlight but have been reviewed elsewhere. 21,22…”