Characterization of the Actinonin Biosynthetic Gene Cluster

Wolf F, Leipoldt F, Kulik A, Wibberg D, Kalinowski J, Kaysser L. Characterization of the Actinonin Biosynthetic Gene Cluster. CHEMBIOCHEM. 2018;19(11):1189-1195.The hydroxamate moiety of the natural product actinonin mediates inhibition of metalloproteinases because of its chelating properties towards divalent cations in the active site of those enzymes. Owing to its antimicrobial activity, actinonin has served as a lead compound for the development of new antibiotic drug candidates. Recently, we identified a putative gene cluster for the biosynthesis of actinonin. Here, we confirm and characterize this cluster by heterologous pathway expression and gene-deletion experiments. We assigned the biosynthetic gene cluster to actinonin production and determine the cluster boundaries. Furthermore, we establish that ActI, an AurF-like oxygenase, is responsible for the N-hydroxylation reaction that forms the hydroxamate warhead. Our findings provide the basis for more detailed investigations of actinonin biosynthesis

Diversity of ABBA Prenyltransferases in Marine Streptomyces sp. CNQ-509: Promiscuous Enzymes for the Biosynthesis of Mixed Terpenoid Compounds.

Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 -CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325) and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene)-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications

Structural basis for non-genuine phenolic acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase CloQ from the ABBA/PT-barrel superfamily.

Acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase SrCloQ was investigated using different non-genuine phenolic compounds. RP-UHPLC-UV-MSn was used for the tentative annotation and quantification of the prenylated products. Flavonoids, isoflavonoids and stilbenoids with different types of substitution were prenylated by SrCloQ, although with less efficiency than the genuine substrate 4-hydroxyphenylpyruvate. The isoflavan equol, followed by the flavone 7,4'-dihydroxyflavone, were the best non-genuine acceptor substrates. B-ring C-prenylation was in general preferred over A-ring C-prenylation (ratio 5:1). Docking studies of non-genuine acceptor substrates with the B-ring oriented towards the donor substrate dimethylallyl pyrophosphate, showed that the carbonyl group of the C-ring was able to make stabilizing interactions with the residue Arg160, which might determine the preference observed for B-ring prenylation. No reaction products were formed when the acceptor substrate had no phenolic hydroxyl groups. This preference can be explained by the essential hydrogen bond needed between a phenolic hydroxyl group and the residue Glu281. Acceptor substrates with an additional hydroxyl group at the C3' position (B-ring), were mainly O3'-prenylated (> 80% of the reaction products). This can be explained by the proximity of the C3' hydroxyl group to the donor substrate at the catalytic site. Flavones were preferred over isoflavones by SrCloQ. Docking studies suggested that the orientation of the B-ring and of the phenolic hydroxyl group at position C7 (A-ring) of flavones towards the residue Tyr233 plays an important role in this observed preference. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family

Complete Genome Sequence of Streptomyces sp. CNQ-509, a Prolific Producer of Meroterpenoid Chemistry

Rückert C, Leipoldt F, Zeyhle P, et al. Complete Genome Sequence of Streptomyces sp. CNQ-509, a Prolific Producer of Meroterpenoid Chemistry. Journal of Biotechnology. 2015;216:140-141.: Streptomyces sp. CNQ-509 is a marine actinomycete belonging to the MAR4 streptomycete lineage. MAR4 strains have been linked to the production of diverse and otherwise rare meroterpenoid compounds. The genome sequence of Streptomyces sp. CNQ-509 was found to contain 29 putative gene clusters for the biosynthesis of secondary metabolites, some of them potentially involved in the formation of meroterpenoid molecules

Phylogenetic tree of ABBA prenyltransferases of the phenol / phenazine family.

<p>Data include previously biochemically characterised ABBA prenyltransferases and those investigated in this study. The tree was constructed with MEGA6 using default parameter for multiple sequence alignment (CLUSTALW) and neighbour-joining method. Bootstrap values (in percent) calculated from 1000 replications are shown at the respective nodes. The fungal indole prenyltransferase DMATS (shares PT barrel) serves as a root.</p

Mixed-terpenoid secondary metabolites of <i>Streptomyces</i> sp. CNQ-509.

<p>Mixed-terpenoid secondary metabolites of <i>Streptomyces</i> sp. CNQ-509.</p

<i>Sr</i>CloQ reaction products tentatively annotated by RP-UHPLC-UV-MSⁿ.

<p><i>Sr</i>CloQ reaction products tentatively annotated by RP-UHPLC-UV-MSⁿ.</p

Structural basis for non-genuine phenolic acceptor substrate specificity of <i>Streptomyces roseochromogenes</i> prenyltransferase CloQ from the ABBA/PT-barrel superfamily

<div><p>Acceptor substrate specificity of <i>Streptomyces roseochromogenes</i> prenyltransferase SrCloQ was investigated using different non-genuine phenolic compounds. RP-UHPLC-UV-MSn was used for the tentative annotation and quantification of the prenylated products. Flavonoids, isoflavonoids and stilbenoids with different types of substitution were prenylated by SrCloQ, although with less efficiency than the genuine substrate 4-hydroxyphenylpyruvate. The isoflavan equol, followed by the flavone 7,4’-dihydroxyflavone, were the best non-genuine acceptor substrates. B-ring <i>C</i>-prenylation was in general preferred over A-ring <i>C</i>-prenylation (ratio 5:1). Docking studies of non-genuine acceptor substrates with the B-ring oriented towards the donor substrate dimethylallyl pyrophosphate, showed that the carbonyl group of the C-ring was able to make stabilizing interactions with the residue Arg160, which might determine the preference observed for B-ring prenylation. No reaction products were formed when the acceptor substrate had no phenolic hydroxyl groups. This preference can be explained by the essential hydrogen bond needed between a phenolic hydroxyl group and the residue Glu281. Acceptor substrates with an additional hydroxyl group at the <i>C</i>3’ position (B-ring), were mainly <i>O</i>3’-prenylated (> 80% of the reaction products). This can be explained by the proximity of the C3’ hydroxyl group to the donor substrate at the catalytic site. Flavones were preferred over isoflavones by SrCloQ. Docking studies suggested that the orientation of the B-ring and of the phenolic hydroxyl group at position <i>C</i>7 (A-ring) of flavones towards the residue Tyr233 plays an important role in this observed preference. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family.</p></div