The Rieske Oxygenase SnoT Catalyzes 2′′‐Hydroxylation of l‐Rhodosamine in Nogalamycin Biosynthesis

Abstract

Nogalamycin is an anthracycline anti-cancer agent that intercalates into the DNA double helix. The binding is facilitated by two carbohydrate units, l-nogalose and l-nogalamine, that interact with the minor and major grooves of DNA, respectively. However, recent investigations have shown that nogalamycin biosynthesis proceeds through the attachment of l-rhodosamine (2''-deoxy-4''-epi-l-nogalamine) to the aglycone. Herein, we demonstrate that the Rieske enzyme SnoT catalyzes 2''hydroxylation of l-rhodosamine as an initial post-glycosylation step. Furthermore, we establish that the reaction order continues with 2-5'' carbocyclization and 4'' epimerization by the non-heme iron and 2-oxoglutarate-dependent enzymes SnoK and SnoN, respectively. These late-stage tailoring steps are important for the bioactivity of nogalamycin due to involvement of the 2''-and 4''-hydroxy groups of l-nogalamine in hydrogen bonding interactions with DNA.

Anthracyclines: biosynthesis, engineering and clinical applications

“…162 In nogalamycin biosynthesis, the Rieske enzyme SnoT performs 2′′-hydroxylation of the l -nogalamine sugar using a [2S–2Fe] iron sulfur cluster after the second glycosylation event. 163 The dual attachment of the aminosugar is completed by the non-heme iron and α-ketoglutarate dependent SnoK (Fig. 8A).…”

Pathway Engineering of Anthracyclines: Blazing Trails in Natural Product Glycodiversification

“…Nonetheless, the pathway intermediates isolated from S. albus typically contained lrhodosamine (2"-deoxy-4"-epi-l-nogalamine) as the carbohydrate unit. Recently, the Rieske enzyme SnoT and two non-heme Fe(II) and α-ketoglutarate dependent enzymes SnoK and SnoN are responsible for late-stage modifications in the biosynthesis of l-nogalamine 55,56 . Complementation experiments with snoN in the 14-producing strain resulted in new 4"epimerized nogalamycins: 4"-epi-nogalamycin R ( 17) and 4"-epi-7-denogalosyl-7-O-α-lolivosyl-nogalamycin R (18) (Figure 5).…”

“…Nonetheless, the pathway intermediates isolated from S. albus typically contained l -rhodosamine (2′′-deoxy-4′′- epi - l -nogalamine) as the carbohydrate unit. Recently, the Rieske enzyme SnoT and two nonheme Fe­(II) and α-ketoglutarate dependent enzymes SnoK and SnoN are responsible for late-stage modifications in the biosynthesis of l -nogalamine. , Complementation experiments with snoN in the 14 -producing strain resulted in new 4″-epimerized nogalamycins: 4″- epi -nogalamycin R ( 17 ) and 4″- epi -7-denogalosyl-7- O -α- l -olivosyl-nogalamycin R ( 18 ) (Figure ). The deletion of snoK from pSnogaORI resulted in the production of new nogalamycins lacking the C5″-2 carbocycle: 10- O -α- l -rhodosamine-nogalamycin F ( 19 ) and 10- O -α- l -rhodosamine-7-denogalosyl-7- O -α- l -olivosyl-nogalamycin F (Figure ) ( 20 ).…”

Mechanism of Two-Component Mono-Oxygenases Involved in Anthracycline 1-Hydroxylation

“…In order to harness the use of O 2 in biochemistry, nature has evolved several classes of enzymes that utilize organic cofactors such as flavin or transition metals to break the spin barrier-limiting reactivity . Examples of classical mono-oxygenases or oxidases involved in anthracycline biosynthesis include flavoenzymes such as the 11-hydroxylase RdmE residing on the rhodomycin pathway, the cytochrome P450 enzyme DoxA responsible for several oxidations in daunorubicin biosynthesis, the 2-oxoketoglutarate and nonheme iron-dependent carbocyclase SnoK, and the Rieske mono-oxygenase SnoT involved in nogalamycin ( 3 ) biosynthesis (Figure a).…”