Abstract
Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C–H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions. We present high resolution crystal structures of SxtT and GxtA with the native β-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site. Ultimately, this structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C–H hydroxylation event. Substitution of these residues produces a SxtT variant that is fully adapted to exhibit the non-native site-selectivity and substrate scope of GxtA. Importantly, we also found that these selectivity regions are conserved in other structurally characterized Rieske oxygenases, providing a framework for predictively repurposing and manipulating Rieske oxygenases as biocatalysts.
Highlights
Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C–H bond functionalization reactions
Unlike what is suggested by the traditional lock–key model of enzyme catalysis, which emphasizes only the interactions formed between the substrate and the active site, the so-called “keyhole”, or auxiliary tunnel and loop regions that interact with a substrate, are emerging as enzyme engineering hotspots, mainly recognized for their potential influence on protein stability, substrate scope, and reaction selectivity (Fig. 1c)[30,31,36,40,41]
We elaborated on the design principles that dictate how the Rieske oxygenases SxtT and GxtA catalyze site-selective monohydroxylation reactions
Summary
Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C–H bond functionalization reactions. We present high resolution crystal structures of SxtT and GxtA with the native β-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site This structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C–H hydroxylation event. To investigate the architectural parameters that dictate the substrate specificity and site-selectivity of a Rieske oxygenase catalyzed reaction, we focused on two Rieske oxygenases, SxtT and GxtA, which are involved in the biosynthesis of paralytic shellfish toxins These enzymes share 88% sequence identity with one another and selectively install a hydroxyl group on adjacent C12 and C11 positions of a tricyclic saxitoxin scaffold, respectively (Fig. 1d, e and Supplementary Fig. 1)[42]. This work outlines the design principles for engineering Rieske oxygenases to have improved activity, broader substrate scope, or altered reaction specificity
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