Enzymatic C_β–H Functionalization of l-Arg and l-Leu in Nonribosomally Derived Peptidyl Natural Products: A Tale of Two Oxidoreductases

Abstract

Muraymycins are peptidyl nucleoside antibiotics that contain two Cβ-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-β-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors thatlike muraymycinare derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by Cβ-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe2+- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe2+- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific Cβ hydroxylation of the Leu residue to generate (2S,3S)-β-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-Cβ-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.

Characterization and Structural Determination of CmnG‐A, the Adenylation Domain That Activates the Nonproteinogenic Amino Acid Capreomycidine in Capreomycin Biosynthesis

“…This study, as well as previous ones, provides evidence that l ‐Cap is synthesized from l ‐Arg before incorporation into the NRPS machinery in CMN and VIO biosynthesis [13] . By contrast, the l ‐ epi ‐Cap moiety is synthesized after the precursor l ‐Arg incorporated into the NRPS machinery in muraymycin biosynthesis, where an unusual dehydrogenase catalyzes C β ‐N bond formation of l ‐Arg to form l ‐ epi ‐Cap [16] . The A domain of VioG (VioG‐A) in VIO biosynthesis was reported to specifically activates l ‐Cap [13] .…”

“…[13] By contrast, the l-epi-Cap moiety is synthesized after the precursor l-Arg incorporated into the NRPS machinery in muraymycin biosynthesis, where an unusual dehydrogenase catalyzes C β -N bond formation of l-Arg to form l-epi-Cap. [16] The A domain of VioG (VioG-A) in VIO biosynthesis was reported to specifically activates l-Cap. [13] CmnG-A and VioG-A share ~59 % amino acid sequence identity (Figure S9) and their 10-residue substrate-specificity sequences are highly conserved (90 %, Table 2).…”

“…Ten-residue substrate-specificity sequences in selected A domains, DhbE, [30] VinN, [35] PheA, [28] SrfA-C, [36] DltA, [37] VioG-A, [13] and Mur12. [15,16] ChemBioChem crystal structures of CmnG-A also serve as a basis for future enzyme engineering for the development of new NRP compounds via incorporation of alternative amino acids.…”

Structural diversity, biosynthesis, and biological functions of lipopeptides fromStreptomyces

“…The peptide scaffold of the muraymycins is assembled by NRPS enzymes (Mur12-Mur14, Mur21, Mur25 and Mur27). Recently, Mur22 was characterised as an FAD-dependent dehydrogenase: a cyclase that generates the epicapreomycidine residue via dehydrogenation and intramolecular cyclisation of L-Arg, while Mur15 was conrmed as a nonheme Fe 2+ -and aKG-dependent dioxygenase, generating 239 Interestingly, modications of L-Arg and Leu occur aer NRPS-mediated peptide chain extension. Since no lipase was found in the mur gene cluster, addition of the FA remains unknown.…”

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

“…Recent advances have shown that capreomycidines can be divergently biosynthesized on NRPSs via dehydrogenation and thioester-mediated Michael addition reactions in the faulknamycin and muraymycin biosynthetic pathways. 10,11 Currently, cyclic arginine ncAAs have limited commercial availability due to their multiple synthetic steps and heavy reliance on protecting groups. 2 This has hampered an understanding of their role in establishing the bioactivities of their cognate NRPS products.…”