Mining Translation Inhibitors by a Unique Peptidyl-Aminonucleoside Synthetase Reveals Cystocin Biosynthesis and Self-Resistance

• Published in: International journal of molecular sciences 2024-12, Vol.25 (23), p.12901
• Main Authors: Alferova, Vera A, Zotova, Polina A, Baranova, Anna A, Guglya, Elena B, Belozerova, Olga A, Pipiya, Sofiya O, Kudzhaev, Arsen M, Logunov, Stepan E, Prokopenko, Yuri A, Marenkova, Elisaveta A, Marina, Valeriya I, Novikova, Evgenia A, Komarova, Ekaterina S, Starodumova, Irina P, Bueva, Olga V, Evtushenko, Lyudmila I, Ariskina, Elena V, Kovalchuk, Sergey I, Mineev, Konstantin S, Babenko, Vladislav V, Sergiev, Petr V, Lukianov, Dmitrii A, Terekhov, Stanislav S
• Format: Article
• Language: English
• Subjects: Amino acids; Analysis; Anti-Bacterial Agents - biosynthesis; Anti-Bacterial Agents - pharmacology; Antibiotics; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biosynthesis; Cells; Cysteine; Dehydrogenases; Drug resistance in microorganisms; Enzymes; Genetic engineering; Genetically modified organisms; Genomes; Genomics; Humans; Metabolites; Mines and mineral resources; Multigene Family; Peptide Synthases - genetics; Peptide Synthases - metabolism; Peptides; Phylogenetics; Protein Biosynthesis; Protein synthesis; Protein Synthesis Inhibitors - metabolism; Protein Synthesis Inhibitors - pharmacology; Proteins; Puromycin - metabolism; Puromycin - pharmacology; Ribonucleic acid; Rifamycins; RNA; Streptomyces - genetics; Streptomyces - metabolism; Transfer RNA
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms252312901

Puromycin (Puro) is a natural aminonucleoside antibiotic that inhibits protein synthesis by its incorporation into elongating peptide chains. The unique mechanism of Puro finds diverse applications in molecular biology, including the selection of genetically engineered cell lines, in situ protein synthesis monitoring, and studying ribosome functions. However, the key step of Puro biosynthesis remains enigmatic. In this work, pur6-guided genome mining is carried out to explore the natural diversity of Puro-like antibiotics. The diversity of biosynthetic gene cluster (BGC) architectures suggests the existence of distinct structural analogs of puromycin encoded by pur-like clusters. Moreover, the presence of tRNA in some BGCs, i.e., -like clusters, leads us to the hypothesis that Pur6 utilizes aminoacylated tRNA as an activated peptidyl precursor, resulting in cysteine-based analogs. Detailed metabolomic analysis of sp. VKM Ac-502 containing -like BGC revealed the production of a cysteinyl-based analog of Puro-cystocin (Cst). Similar to puromycin, cystocin inhibits both prokaryotic and eukaryotic translation by the same mechanism. Aminonucleoside N-acetyltransferase CstC inactivated Cst, mediating antibiotic resistance in genetically modified bacteria and human cells. The substrate specificity of CstC originated from the steric hindrance of its active site. We believe that novel aminonucleosides and their inactivating enzymes can be developed through the directed evolution of the discovered biosynthetic machinery.