8 research outputs found
A ThDP-dependent enzymatic carboligation reaction involved in Neocarazostatin A tricyclic carbazole formation
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31570033 to Y. Y.) and the Leverhulme Trust-Royal Society Africa Award (AA090088 to K. K and H. D.). Open access via RSC Gold 4 Gold.Peer reviewedPublisher PD
Characterization of the Biosynthetic Gene Cluster for Benzoxazole Antibiotics A33853 Reveals Unusual Assembly Logic
SummaryA33853, which shows excellent bioactivity against Leishmania, is a benzoxazole-family compound formed from two moieties of 3-hydroxyanthranilic acid and one 3-hydroxypicolinic acid. In this study, we have identified the gene cluster responsible for the biosynthesis of A33853 in Streptomyces sp. NRRL12068 through genome mining and heterologous expression. Bioinformatics analysis and functional characterization of the orfs contained in the gene cluster revealed that the biosynthesis of A33853 is directed by a group of unusual enzymes. In particular, BomK, annotated as a ketosynthase, was found to catalyze the amide bond formation between 3-hydroxypicolinic and 3-hydroxyanthranilic acid during the assembly of A33853. BomJ, a putative ATP-dependent coenzyme A ligase, and BomN, a putative amidohydrolase, were further proposed to be involved in the benzoxazole formation in A33853 according to gene deletion experiments. Finally, we have successfully utilized mutasynthesis to generate two analogs of A33853, which were reported previously to possess excellent anti-leishmanial activity
Biosynthesis of Neocarazostatin A Reveals the Sequential Carbazole Prenylation and Hydroxylation in the Tailoring Steps
Highlights
ā¢ The biosynthetic gene cluster of neocarazostatin A was identified
ā¢ A new type of carbazole prenyltransferases, NzsG, was characterized
ā¢ The P450 enzyme NzsA catalyzing the last step of the biosynthesis was identified
ā¢ The biotransformation in the late stage of the biosynthesis was reconstituted
Summary
Neocarazostatin A (NZS) is a bacterial alkaloid with promising bioactivities against free radicals, featuring a tricyclic carbazole nucleus with a prenyl moiety at C-6 of the carbazole ring. Here, we report the discovery and characterization of the biosynthetic pathway of NZS through genome mining and gene inactivation. The in vitro assays characterized two enzymes: NzsA is a P450 hydroxylase and NzsG is a new phytoene-synthase-like prenyltransferase (PTase). This is the first reported native PTase that specifically acts on the carbazole nucleus. Finally, our in vitro reconstituted experiment demonstrated a coupled reaction catalyzed by NzsG and NzsA tailoring the NZS biosynthesis
A ThDP-dependent enzymatic carboligation reaction involved in Neocarazostatin A tricyclic carbazole formation
Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis
Apramycin is a clinically
interesting aminoglycoside antibiotic
(AGA) containing a highly unique bicyclic octose moiety, and this
octose is deoxygenated at the C3 position. Although the biosynthetic
pathways for most 2-deoxystreptamine-containing AGAs have been well
characterized, the pathway for apramycin biosynthesis, including the
C3 deoxygenation process, has long remained unknown. Here we report
detailed investigation of apramycin biosynthesis by a series of genetic,
biochemical and bioinformatical studies. We show that AprD4 is a novel
radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme,
which uses a noncanonical CX<sub>3</sub>CX<sub>3</sub>C motif for
binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine,
a pseudodisaccharide intermediate in apramycin biosynthesis. We also
show that AprD3 is an NADPH-dependent reductase that catalyzes the
reduction of the dehydrated product from AprD4-catalyzed reaction
to generate lividamine, a C3ā² deoxygenated product of paromamine.
AprD4 and AprD3 do not form a tight catalytic complex, as shown by
protein complex immunoprecipitation and other assays. The AprD4/AprD3
enzyme system acts on different pseudodisaccharide substrates but
does not catalyze the deoxygenation of oxyapramycin, an apramycin
analogue containing a C3 hydroxyl group on the octose moiety, suggesting
that oxyapramycin and apramycin are partitioned into two parallel
pathways at an early biosynthetic stage. Functional dissection of
the C6 dehydrogenase AprQ shows the crosstalk between different AGA
biosynthetic gene clusters from the apramycin producer <i>Streptomyces
tenebrarius</i>, and reveals the remarkable catalytic versatility
of AprQ. Our study highlights the intriguing chemistry in apramycin
biosynthesis and natureās ingenuity in combinatorial biosynthesis
of natural products