Evolution‐guided engineering of non‐heme iron enzymes involved in nogalamycin biosynthesis

Abstract

Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. The key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison with their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to nonheme iron and 2‐oxoglutarate‐dependent mono‐oxygenases. In spite of structural similarity, the two proteins catalyze distinct chemical reactions; SnoK is a C2–C5″ carbocyclase, whereas SnoN catalyzes stereoinversion at the adjacent C4″ position. Here, we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyze stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C‐terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for the formation of the C2–C5″ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents.EnzymesEC number 1.14.11.

Pathway Engineering of Anthracyclines: Blazing Trails in Natural Product Glycodiversification

“…This was demonstrated in a recent study where SnoN was engineered to catalyze carbocyclization after insertion of just three residues from SnoK. 57 2.3. Keyicin.…”

“…Despite the drastically different chemistry catalyzed by SnoK and SnoN, the catalytic difference appears to depend largely on the positioning of the substrates in front of the activated iron-oxo center. This was demonstrated in a recent study where SnoN was engineered to catalyze carbocyclization after insertion of just three residues from SnoK …”

Anthracyclines: biosynthesis, engineering and clinical applications

“…8A), which surprisingly shares signicant sequence similarity with SnoK despite the difference in enzymatic activity. 164,165 Other unique post-PKS tailoring steps include oxidation and methylation reactions by P450 and SAM-dependent proteins, respectively, on the steffimycin pathway. 124 An atypical oxidoreductase AknOx catalyzes two subsequent oxidations of the terminal L-rhodosamine sugar to L-aculose supported by a bicovalently bound FAD on the aclacinomycin pathway.…”

“…8A), which surprisingly shares significant sequence similarity with SnoK despite the difference in enzymatic activity. 164,165…”