Unravelling key enzymatic steps in C-ring cleavage during angucycline biosynthesis

Abstract

Angucyclines are type II polyketide natural products, often characterized by unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone. While the enzymes involved in B-ring cleavage have been extensively studied, little is known of the enzymes leading to C-ring cleavage. Here, we unravel the function of the oxygenases involved in the biosynthesis of lugdunomycin, a highly rearranged C-ring cleaved angucycline derivative. Targeted deletion of the oxygenase genes, in combination with molecular networking and structural elucidation, showed that LugOI is essential for C12 oxidation and maintaining a keto group at C6 that is reduced by LugOII, resulting in a key intermediate towards C-ring cleavage. An epoxide group is then inserted by LugOIII, and stabilized by the novel enzyme LugOV for the subsequent cleavage. Thus, for the first time we describe the oxidative enzymatic steps that form the basis for a wide range of rearranged angucycline natural products.

Divergence of Classical and C-Ring-Cleaved Angucyclines: Elucidation of Early Tailoring Steps in Lugdunomycin and Thioangucycline Biosynthesis

“…QL37, which confirms that the proteins are orthologous, and justifies the replacement of LugOI with PgaE in our assays. 31 The combination of PgaE and LugOIIred converted 8 into 9 (Figure 2A) similarly to PgaE and LanV, 20 which indicated that LugOIIred is orthologous to LanV in terms of substrate specificity and catalytic activity; both enzymes prefer the 12hydroxylated derivative of 8 as a substrate and catalyze 6ketoreduction leading to 6R configuration. This was in contrast to UrdMred from the urdamycin pathway, which utilizes a 12,12b-dihydroxylated substrate and converts 8 to 2 with 6S configuration in the presence of PgaE (Figure 2A), as we have previously reported.…”

“…However, lugOIV and tacV genes may be redundant, as their deletion did not substantially change the production profiles of the strains. 29,31 Enzymes of the ABM family, such as LugOIII/TacS and LugOV/TacT, which have been implicated in 6a/12a 1B) from the jadomycin pathway. 12,28 No homologous proteins were present in classical or A-ring-cleaved angucycline BGCs with the exception of the hatomarubigin BGC 34 encoded HrbF, which has 44% sequence identity with LugOV (Table 1).…”

“…The novel reaction products 18 and 19 were identified using mass spectrometry (MS) (Figures S49 and S50), which revealed 2 Da higher masses for the products in comparison to the respective substrates, which is consistent with the 1-ketoreduction activity previously confirmed for 16 and 17. 27 The possibility of ketoreduction at positions 7 and 12 for 18 and 19 were ruled out based on their UV−vis spectra (Figure S57), which did not 31 To confirm the timing of 8-O-methylation and the order of the tailoring reactions, we performed a series of coupled reactions using 7 as a substrate (Figure 3). The incubation of 7 with the SAM-dependent 8-O-methyltransferase LugN alone did not lead to enzymatic turnover, except for conversion of trace impurity 15 to methylated derivative 13.…”

“…In lugdunomycin biosynthesis, the 6-ketoreduction activity of the SDR-family enzyme LugOIIred has been demonstrated in vivo, but remarkably, the enzyme has been shown to additionally catalyze 1-ketoreduction of 8- O -methyltetrangomycin ( 12 ) and 8- O -methylrabelomycin ( 13 ) in vitro . Very recently, the ABM family genes lugOIII and lugOV have been identified to catalyze 6a/12a epoxidation and implicated in C-ring cleavage, respectively, based on in vivo data . Metabolic in vivo studies into the biosynthesis of thioangucycline have implicated the SDR-family enzymes TacA and TacO to catalyze the reduction of the quinone carbonyl groups at positions 12 and 7, respectively .…”

“…The co-occurrence of quinone reduction and 6a/12a epoxidation in other C-ring-cleaved angucyclinones such as rubiginone H is also noteworthy. Hence, the reduction of the C-ring has been suggested to be involved in C-ring cleavage through Grob-type fragmentation of the 6a,12a-epoxide. ,− …”