Structural Insight into the Catalytic Mechanism of Non-Heme Iron Halogenase AdaV in 2′-Chloropentostatin Biosynthesis

Non-heme Fe2+ and 2-oxoglutarate (Fe2+/2OG)-dependent halogenases are a promising platform for biocatalytic halogenations owing to their ability to functionalize unactivated sp(3) C-H bonds. To date, however, relatively few Fe2+/2OG-dependent halogenases have been identified that act on small stand-alone molecules. AdaV, a member of the carrier-protein-independent halogenases, selectively modifies the unactivated C2 ' of the dAMP in 2 '-chloropentostatin biosynthesis. In this study, we report the X-ray crystallographic structures of the AdaV complex with its substrate and various AdaV variants. The combined crystallographic and biochemical data clarify the molecular mechanism of AdaV for its substrate specificity and stereoselectivity. Moreover, we have engineered the AdaV(Q203A)/AdaV(V269A) variant to produce a mixture of halogenated and hydroxylated products and further engineered AdaV(Q203A&V269A&G196D/E) variants to merely keep hydroxylation activity. Remarkably, we have also proposed a dual-controlling mechanism for AdaV catalysis, in which G196 plays an important role in halogenation by creating an iron coordination site for chloride binding, while Q203&V269 serve to orient the Fe(III)-OH intermediate to constrain the oxygen rebounding onto the radical substrate. These results greatly expand the enzymatic repertoire regarding halogenated natural product biosynthesis and open the way for the rational and rapid discovery of more AdaV-related enzymes as toolkits for further synthetic biology uses.