Catalysis of Extracellular Deamination by a FAD‐Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin A

Abstract

The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process,atthe end of which the matured compounds are exported outside the cells.T he biosynthesis of saframycin A( SFM-A), an antitumor antibiotic,r equires ac ryptic fatty acyl chain to guide the construction of ap entacyclic tetrahydroisoquinoline scaffold; however,t he follow-up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, am embrane-bound peptidase,h ydrolyzes the fatty acyl chain to release the amino group;and SfmCy2, asecreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly.T hese results not only fill in the missing steps of SFM-A biosynthesis,but also reveal that aF AD-binding oxidoreductase catalyzesa nu nexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.

Reductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics

“…We have been pursuing the biosynthetic studies of SFM-A (4, Fig. 1a) [24][25][26][27] ; therefore, the sfm BGC and the genome of SFM-A producer, S. lavendulae NRRL 11002, were carefully analyzed to search for genes encoding NapW-like SDRs. Indeed, in addition to SfmO1 (56% identity with NapW) encoded by sfmO1 within sfm BGC, there are two another homologous proteins homO1a (62% identity) and homO1b (60% identity) encoded by genes outside the BGC.…”

Modification of ɛ‐poly‐L‐lysine in vivo to reduce self‐toxicity and enhance antibiotic overproduction

“…This weaker inhibitory effect of 2‐PL‐G on its producing strain may be a resistance mechanism of S. albulus J1–005 against ɛ‐PL. These esterified derivatives could be released into culture broth, which differs from the prodrug mechanism of resistance in which removal of the modification occurs during secretion outside across the membrane …”

Extracellularly oxidative activation and inactivation of matured prodrug for cryptic self-resistance in naphthyridinomycin biosynthesis

“…Previous studies have established that the Tat system is a major and general route of protein export in S. coelicolor (26). Additionally, we recently identified a secreted oxidoreductase SfmCy2 bearing a similar Tat signal peptide, which catalyzes an oxidative deamination extracellularly in saframycin A biosynthesis from S. lavendulae (27). These collective evidences facilitate us to speculate that: (i) NapU might be a secreted oxidase to catalyze an extracellular hydroxylation reaction; (ii) the two bands in SDS/PAGE should be the full and N signal-cleaved variants.…”

“…General materials, gene inactivation/complementation, fermentation, production and analysis of NDM related intermediates, protein expression, and purification are all following previous methods (19,20,27) except using different plasmids, strains (SI Appendix, Table S1), and PCR primers (SI Appendix, Table S2). Additional SI includes three tables (SI Appendix, Tables S1-S3) extract.…”