Rieske Non-heme Iron Oxygenases in Natural Product Biosynthesis

Abstract

Nature has evolved a striking array of enzymes that perform precise C-H bond functionalization reactions. Rieske oxygenases represent one of the most prevalent and underexploited enzyme classes that falls in this category. Rieske oxygenases are enzymes that couple a 2-His/2-Cys ligated [2Fe-2S] cluster, or Rieske cluster, with a mononuclear iron site. The Rieske cluster accepts electrons and shuttles them to a mononuclear iron site to catalyze the desired reaction. Some enzymes in this class function as dioxygenases and incorporate both oxygen atoms of O2 into a product, whereas others function as monooxygenases and insert only one oxygen atom into a product. Remarkably less well studied, there is even one enzyme in this class that is proposed to convert a methyl group into a formyl group via sequential mono-oxygenation reactions. Despite the ubiquity of these enzymes in Nature and their promise for producing pharmaceuticals, commodity chemicals, and facilitating bioremediation efforts, there is still a critical gap in knowledge with regards to the structure-function relationships in this class of enzymes, leaving many open questions about how these enzymes catalyze reactions selectively or catalyze reactions with different outcomes. Saxitoxin and Chlorophyll are two natural products that are biosynthesized, in part, by Rieske oxygenases. Saxitoxin is a well-known paralytic shellfish toxin and Chlorophyll is one of the most significant pigments in photosynthesis. Two Rieske non-heme iron oxygenases, SxtT and GxtA, involved in saxitoxin biosynthesis are discussed in chapters 2 and 3. These two Rieske oxygenases share 88-pecent sequence identity with one another but catalyze hydroxylation reactions on the saxitoxin scaffold at different positions. Using X-ray crystallography, rigorous structural analysis, and biochemical studies, we revealed the design principle for site-selective hydroxylation by SxtT and GxtA. Through analysis of the structurally available enzymes, we further showed that these design principles may be universally conserved in the Rieske oxygenase class. Another interesting Rieske oxygenase studied here is involved in Chlorophyll biosynthesis and is discussed in chapter 4. This enzyme, Chlorophyll(ide) a oxygenase (CAO), is a key player in the Chlorophyll cycle and is proposed to transform Chlorophyll(ide) a into Chlorophyll(ide) b by catalyzing two sequential monooxygenation reactions that convert a C7-methyl group into a C7-formyl group. In this work, using a bottom-up approach, we overproduced and reconstituted the activity of CAO from four different organisms from different kingdoms of life. Importantly, this work demonstrates that CAO converts a Chlorophyll precursor, Chlorophyllide a, into Chlorophyllide b in vitro. We established the existence of a monooxygenated intermediate, supporting the proposal that CAO is a Rieske oxygenase which catalyzes two iterative monooxygenation reactions. We revealed key details about the stereoselectivity of this reaction and the substrate scope of CAO. Finally, through the enzymatic synthesis of an unnatural Chlorophyll pigment, we demonstrated the potential for using CAO as an enzymatic tool for synthesizing custom-tuned chlorophyll pigments. Taken together, this research adds to our fundamental understanding of how saxitoxin and Chlorophyll are biosynthesized. In addition, these studies reveal predictive power for thinking about how to control selectivity and redesign reactivity of Rieske oxygenases.