Saframycin A

The antitumor tetrahydroisoquinoline (THIQ) alkaloids share a common pentacyclic scaffold that is biosynthesized by nonribosomal peptide synthetases involving unique enzymatic Pictet-Spengler cyclizations. Herein we report concise and divergent chemo-enzymatic total syntheses of THIQ alkaloids by merging precise chemical synthesis with in vitro engineered biosynthesis. A recombinant enzyme SfmC responsible for the biosynthesis of saframycin A was adapted for the assembly of these natural products and their derivatives, by optimizing designer substrates compatible with SfmC through chemical synthesis. The appropriately functionalized pentacyclic skeleton were efficiently synthesized by streamlining the linkage between SfmC-catalyzed multistep enzymatic conversions and chemical manipulations of the intermediates to install aminonitrile and N-methyl groups. This approach allowed rapid access to the elaborated pentacyclic skeleton in a single day starting from two simple synthetic substrates without isolation of the intermediates. Further functional group manipulations allowed operationally simple and expeditious syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3 that could be versatile and common precursors for the artificial production of other antitumor THIQ alkaloids and their variants.

The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process, at the end of which the matured compounds are exported outside the cells. The biosynthesis of saframycin A (SFM-A), an antitumor antibiotic, requires a cryptic fatty acyl chain to guide the construction of a pentacyclic tetrahydroisoquinoline scaffold; however, the follow-up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, a membrane-bound peptidase, hydrolyzes the fatty acyl chain to release the amino group; and SfmCy2, a secreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly. These results not only fill in the missing steps of SFM-A biosynthesis, but also reveal that a FAD-binding oxidoreductase catalyzes an unexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.

Saframycin A (SFM-A) is a potent antitumor antibiotic that belongs to the tetrahydroisoquinoline family. Biosynthetic studies have revealed that its unique pentacyclic core structure is derived from alanine, glycine, and non-proteinogenic amino acid 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5-Me-OMe-Tyr). SfmD, a hypothetical protein in the biosynthetic pathway of SFM-A, was hypothesized to be responsible for the generation of the 3-hydroxy group of 3-OH-5-Me-OMe-Tyr based on previously heterologous expression results. We now report the in vitro characterization of SfmD as a novel heme-containing peroxidase that catalyzes the hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine using hydrogen peroxide as the oxidant. In addition, we elucidated the biosynthetic pathway of 3-OH-5-Me-OMe-Tyr by kinetic studies of SfmD in combination with biochemical assays of SfmM2, a methyltransferase within the same pathway. Furthermore, SacD, a counterpart of SfmD involved in safracin B biosynthesis, was also characterized as a heme-containing peroxidase, suggesting that SfmD-like heme-containing peroxidases may be commonly involved in the biosynthesis of SFM-A and its analogs. Finally, we found that the conserved motif HXXXC is crucial for heme binding using comparative UV-Vis and Magnetic Circular Dichroism (MCD) spectra studies of SfmD wild-type and mutants. Together, these findings expand the category of heme-containing peroxidases and set the stage for further mechanistic studies. In addition, this study has critical implications for delineating the biosynthetic pathway of other related tetrahydroisoquinoline family members.