Enzymatic Tailoring in Luzopeptin Biosynthesis Involves Cytochrome P450‐Mediated Carbon–Nitrogen Bond Desaturation for Hydrazone Formation

Abstract

Luzopeptins and related decadepsipeptides are bisintercalator nonribosomal peptides featuring rare acylsubstituted tetrahydropyridazine-3-carboxylic acid (Thp) subunits that are critical to their biological activities.H erein, we reconstitute the biosynthetic tailoring pathwayinluzopeptin A biosynthesis through in vivo genetic and in vitro biochemical approaches.S ignificantly,w er evealed am ultitasking cytochrome P450 enzyme that catalyzesf our consecutive oxidations including the highly unusual carbon-nitrogen bond desaturation, forming the hydrazone-bearing 4-OH-Thp residues.M oreover,w ei dentified am embrane-bound acyltransferase that likely mediates the subsequent O-acetylation extracellularly,a sapotential self-protective strategy for the producer strain. Further genome mining of novel decadepsipeptides and an associated P450 enzyme have provided mechanistic insights into the P450-mediated carbon-nitrogen bond desaturation. Our results not only reveal the molecular basis of pharmacophore formation in bisintercalator decadepsipeptides,b ut also expand the catalytic versatility of P450 family enzymes.

Biosynthesis of Tasikamides via Pathway Coupling and Diazonium-Mediated Hydrazone Formation

“…In contrast to the omnipresence of synthetic hydrazones, only a small number of naturally occurring hydrazones are known today (Scheme S1). Several low-molecular-weight hydrazones, such as farylhydrazones and prolinimines, were isolated from fungi, plants, and other eukaryotes as early as 1967. , Hydrazones are also known to be produced by prokaryotes as exemplified by the Streptomyces-produced yoropyrazone, katorazone, S56-p1, and luzopeptins/korkormicins. − The biosynthetic studies on S56-p1 and luzopeptins revealed the formation of hydrazone via the oxidation of hydrazine intermediates, whereas some other natural hydrazones are speculated to be generated by hydrazine–ketone condensation. , …”

“…Surprisingly, the N-H (δ H 10.32) and vinyl methyl (δ H 1.89 and δ C 10.1) signals are reminiscent of the unique arylhydrazone unit in the fungal metabolite farylhydrazones (Scheme S5). 8 The presence of the arylhydrazone functionality, along with the adjacent three-carbon linker (C1−C3), was further substantiated by the 1 H− 15 N HSQC and 1 H− 13 C/ 15 N HMBC correlations (Figure 1C and Figures S2-10 and S2-12). The presence of the unusual arylhydrazone, the hydroxylated and methylated cyclic pentapeptide scaffold, and the lipophilic AHA chain distinguishes 1−3 from other currently known cyclic peptides.…”

“…40−43 Aha1 and Aha2 also share extremely high sequence homology with another pair of S. davawensis enzymes, BN159_4421 (identity/similarity%: 86/90) and BN159_4422 (94/97), whose nitrite-producing enzymatic activity was also confirmed by in vitro assays. 44 When the culture of S. tasikensis P46 was supplemented with 15 NO 2 − , the singly 15 N labeled mass signal ([M + H] + = 1144.5) in 1 was significantly enriched with more than 10-fold in intensity (Figure S16), and the 15 N was found to only incorporate in the N 1 ′-containing fragments on the basis of MS/MS analysis (Scheme S9). Our 1 H− 15 N HMBC experiment further suggested that the 15 N isotope was enriched at the N 1 ′ position (δ N 334.7 ppm) (Figure 4A−C).…”

“…The second pathway synthesizes and diazotizes the alkyl 5-hydroxylanthranilate (AHA) moiety. Unlike the currently known hydrazone-forming mechanisms that involve hydrazine oxidation, , our results suggest a surprising mechanism in which the hydrazone group is formed via enzymatic diazotization for NN bond formation and diazonium-mediated Japp–Klingemann coupling for CN bond formation. The surprising findings indicate that the diversity of naturally occurring hydrazones and hydrazone-generating pathways could be underexplored.…”

“…10−15 The biosynthetic studies on S56-p1 and luzopeptins revealed the formation of hydrazone via the oxidation of hydrazine intermediates, whereas some other natural hydrazones are speculated to be generated by hydrazine−ketone condensation. 13,15 Here we report the discovery of a family of structurally novel cyclic peptides named tasikamides A−G (1−7). Tasikamides A−C (1−3) contain an unexpected hydrazone group that is rarely seen in naturally occurring cyclic peptides.…”