Functional and Structural Insights into a Novel Promiscuous Ketoreductase of the Lugdunomycin Biosynthetic Pathway

Abstract

Angucyclines are a structurally diverse class of actinobacterial natural products defined by their varied polycyclic ring systems, which display a wide range of biological activities. We recently discovered lugdunomycin ( 1 ), a highly rearranged polyketide antibiotic derived from the angucycline backbone that is synthesized via several yet unexplained enzymatic reactions. Here, we show via in vivo , in vitro , and structural analysis that the promiscuous reductase LugOII catalyzes both a C6 and an unprecedented C1 ketoreduction. This then sets the stage for the subsequent C-ring cleavage that is key to the rearranged scaffolds of 1 . The 1.1 Å structures of LugOII in complex with either ligand 8- O -Methylrabelomycin ( 4 ) or 8- O -Methyltetrangomycin ( 5 ) and of apoenzyme were resolved, which revealed a canonical Rossman fold and a remarkable conformational change during substrate capture and release. Mutational analysis uncovered key residues for substrate access, position, and catalysis as well as specific determinants that control its dual functionality. The insights obtained in this work hold promise for the discovery and engineering of other promiscuous reductases that may be harnessed for the generation of novel biocatalysts for chemoenzymatic applications.

Discovery of actinomycin L, a new member of the actinomycin family of antibiotics

“…Samples were analyzed according to the protocol that was previously published 29 . LC–MS/MS acquisition of the pure compounds was performed using Shimadzu Nexera X2 UHPLC system coupled to Shimadzu 9030 QTOF mass spectrometer as previously described 61 . LC–MS/MS acquisition for molecular networking was performed using Thermo Instruments MS system (LTQ Orbitrap XL, Bremen, Germany) equipped with an electrospray ionization source (ESI) as described 29 .…”

A Standalone β-Ketoreductase Acts Concomitantly with Biosynthesis of the Antimycin Scaffold

“…This tetracyclic aromatic compound is significantly different from AntM’s expected substrate, but Patrikainen et al used its positioning and the generation of mutants to identify the R1 to R4 regions in UrdMred and LanV, which were important in determining the substrate specificity and regioselectivity. These regions were also recently shown to be important for another tetracyclic aromatic C6-ketoreductase, LugOII (lugdunomycin) . Sequence alignment of AntM with UrdMred and LanV proteins shows that the R1–R4 regions around the active site are the regions that diverge most between UrdMred/LanV and AntM (Figure C,D), suggesting that these portions of the protein are likely to also determine the substrate specificity of AntM.…”

“…These regions were also recently shown to be important for another tetracyclic aromatic C6-ketoreductase, LugOII (lugdunomycin). 14 Sequence alignment of AntM with UrdMred and LanV proteins shows that the R1−R4 regions around the active site are the regions that diverge most between UrdMred/LanV and AntM (Figure 2C,D), suggesting that these portions of the protein are likely to also determine the substrate specificity of AntM. Interestingly, two of the small side chains that form the rabelomycin binding site in UrdMred (Asn154 and Ile192) and LanV (Ser154 and Ser192) have been replaced by the significantly bulkier side chains of Tyr185 and Phe223 in AntM (Figure 2E).…”

Characterization of the Biosynthetic Gene Cluster and Shunt Products Yields Insights into the Biosynthesis of Balmoralmycin

“…UWM6 is a common intermediate in angucycline biosynthesis and structurally diverse angucyclines can be generated from UWM6 through the action of oxygenases, reductases, and other tailoring-enzymes. 1,11,13,20,23,[60][61][62] Based on the function of their homologs from the landomycin/gilvocarcin/jadomycin pathways, 1,10,63 the FAD-dependent monooxygenase BalA8 could catalyze a 2,3-dehydration reaction to generate the C2=C3 bond. The short-chain dehydrogenase/reductase BalA9, whose homologs can also be found in many angucycline pathways, 20,62 putatively reduces the C-6 keto group to a hydroxyl group.…”

“…The short-chain dehydrogenase/reductase BalA9, whose homologs can also be found in many angucycline pathways, 20,62 putatively reduces the C-6 keto group to a hydroxyl group. Another FAD-dependent monooxygenase (BalA7), which is also conserved in the landomycin/jadomycin pathways, 19,[61][62]64 is predicted to catalyze an oxidation reaction to generate the anthraquinone-type intermediate dehydrorabelomycin (Figure 4). Subsequent glycosylation of dehydrorabelomycin by BalB7 generates the stable intermediate 3 that accumulated in the culture broth.…”