Evolution‐guided engineering of non‐heme iron enzymes involved in nogalamycin biosynthesis. (12th January 2020)

• Record Type: Journal Article
• Authors: Nji Wandi, Benjamin; Siitonen, Vilja; Dinis, Pedro; Vukic, Vladimir; Salminen, Tiina A.; Metsä‐Ketelä, Mikko
• Is Part Of: FEBS journal. Volume 287:Number 14(2020)
• Page Start: 2998
• Page End: 3011
• Publication Date: 2020-01-12
• Subjects: chimeragenesis -- computational modeling -- polyketide -- protein engineering -- Streptomyces; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15192
• Languages: English
• ISSNs: 1742-464X

Abstract

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. Enzymes: EC number 1.14.11 . Abstract : Microbes have the ability to produce libraries of bioactive natural products through their secondary metabolism. The key to this chemical diversity is the rapid functional divergence of biosynthetic enzymes. Here, we have deciphered the evolutionary events leading to the emergence of epimerases from carbocyclases involved in anthracycline biosynthesis. Insight into evolutionary processes of non‐heme iron and 2‐oxoglutarate‐dependent proteins facilitates our understanding how microbes have become a major source of pharmaceuticals.