Abstract
Tricyclic carbazole is an important scaffold in many naturally occurring metabolites, as well as valuable building blocks. Here we report the reconstitution of the ring A formation of the bacterial neocarazostatin A carbazole metabolite. We provide evidence of the involvement of two unusual aromatic polyketide proteins. This finding suggests how new enzymatic activities can be recruited to specific pathways to expand biosynthetic capacities. Finally, we leveraged our bioinformatics survey to identify the untapped capacity of carbazole biosynthesis.
Highlights
Neocarazostatin A 1 is a bacterial metabolite first isolated from the culture of Streptomyces sp
Neocarazostatin A 1 belongs to a group of simple carbazole alkaloids (CAs) with aliphatic side chains
We have delineated the functions of five key enzymes encoded in the biosynthetic gene cluster (BGC) of 1, including the phytoene-synthase-like prenyltransferase NzsG and the P450 hydroxylase NzsA,[1] a thiamine diphosphate-dependent enzyme NzsH,[3] a freestanding acyl carrier protein (ACP) NzsE and a classical βketoacyl−acyl carrier protein synthase III NzsF.[4]
Summary
A similar observation discovered by Kobayashi and coworkers in parallel to our study.[5]. Considering the instability of α-hydroxyl acyloin 3 from NzsH (the structure of 3 has been revised here compared to our previous structural interpretation3), we first performed a one-pot reaction by incubating NzsJ (1 μM) with the enzymatic systems of NzsH and NzsE we’ve established before to generate the acyloin and 3-hydroxybutyryl-NzsE in situ, respectively; two new compounds with mass-to-charge ratios (m/z) of 290.1382 were formed as observed in the LCHR-ESIMS analysis. An enzymatic assay of NzsI with the isolated 5 and 6 resulted in no new product, strongly suggesting that 5 and 6 were rearranged enzymatic products, a similar observation of recent parallel report.[5] This led us to speculate that 4 is the bonafide product of NzsJ that underwent a spontaneous α-ketol rearrangement reaction (Figure 2B). Additional tables of primers, strains, and protein accession numbers; NMR, HR-ESIMS, and UV spectra of reactions and products; sequence analysis of NzsJ and NzsI; DFT computation data and parameters (PDF).
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