Characterization of RufT Thioesterase Domain Reveals Insights into Rufomycin Cyclization and the Biosynthetic Origin...

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Biosynthesis

Background:

• Rufomycins, antimycobacterial nonribosomal cyclic peptides (NRcPs) from *Streptomyces atratus*, are targets for engineering novel derivatives.
• Investigating the thioesterase (TE) domain of non-ribosomal peptide synthetase (NRPS) RufT is crucial for understanding rufomycin macrocyclization and pathway engineering.

Discussion:

• The recombinant RufT-TE domain and RufT-PCP-TE didomain showed limited tolerance to substrate sequence changes, yielding significant hydrolyzed peptide.
• Identification of rufomyazine, a diketopiperazine, suggests non-enzymatic formation from linear peptide products.
• This challenges previous assumptions of solely efficient cyclization and highlights dual product formation.

Key Insights:

• The RufT TE domain produces both cyclic rufomycins and linear peptides.
• Rufomyazine likely forms non-enzymatically from the linear peptide intermediate.
• TE domains may act as gatekeepers in NRPS pathways, controlling product fate.

Outlook:

• Understanding TE domain substrate tolerance is key for rational engineering of NRPS pathways.
• Further studies can elucidate the non-enzymatic diketopiperazine formation mechanism.
• This work provides insights into optimizing rufomycin production and NRPS engineering strategies.