Design principles for site-selective hydroxylation by a Rieske oxygenase

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.

Rieske Oxygenase Catalyzed C–H Bond Functionalization Reactions in Chlorophyll b Biosynthesis

“…All three chiral centers in Chl a , b , and intermediates are labeled with a red asterisk and for the chemical structure, R = H or C 20 H 39 for chlorophyllide and chlorophyll pigments, respectively. This proposal for CAO is unlike that for other transformations that proceed through more than one monooxygenation reaction and require two Rieske oxygenases to be completed. − This proposal is also unlike that needed to convert a methyl group into a formyl group in the catabolism of 4-toluene sulfonate, which requires both a Rieske oxygenase and dehydrogenase . (b) CAO has potential to be used as a tool for formylating the Chl scaffold to produce custom-tuned native and non-native pigments.…”

“…For example, in other biosynthetic pathways where two oxygenation reactions are required, multiple proteins work together (Figure a). The two required oxygenations on the saxitoxin scaffold are made by the Rieske oxygenases SxtT and GxtA (Figure a). − Likewise, two Rieske oxygenases, NdmA and NdmB, catalyze iterative oxidative demethylation reactions in caffeine degradation (Figure a) . The Rieske oxygenase 4-toluenesulfonate methyl monooxygenase (TsaM) also catalyzes a methyl- to formyl-group transformation to convert 4-tolunesulfonate into 4-sulfobenzoate .…”

Engineering Rieske oxygenase activity one piece at a time

“…Based on sequence alignments, this residue appears to be a hallmark of quaternary-ammonium-substrate-oxidizing Rieske oxygenases [ 39 ]. Along these lines, the α 3 Rieske oxygenases SxtT and GxtA, which catalyze monooxygenation reactions at the C12 and C11 positions of a tricyclic saxitoxin scaffold, respectively, use two active site residues to hold the substrate such that the correct carbon-atom is positioned for hydroxylation ( Figure 2f ) [ 37 , 38 , 50 ]. Two important structural studies on CARDO have revealed that mutations in the active site can change the openness of the substrate-binding pocket, the orientation in which the substrate binds, and the outcome of the hydroxylation event ( Figure 2g ) [ 36 , 51 ].…”

Contrasting Mechanisms of Aromatic and Aryl-Methyl Substituent Hydroxylation by the Rieske Monooxygenase Salicylate 5-Hydroxylase

“…Oxygen activation and substrate oxidation occur at the mononuclear iron site with the reduced Rieske cluster [Fe­(II)-Fe­(III)], providing an essential electron for the process. − Despite the many fundamental similarities of the RO enzymes, considerable specificity in substrate selection, reaction type, and regiospecificity is observed for individual family members. X-ray crystal structures of ROs that catalyze a range of reaction types fail to distinguish a basis for this specificity in the structural regions encompassing the Rieske cluster, the residues between the metal centers, and mononuclear iron ligation, which are remarkably conserved. ,,,,− Progress has been made in understanding the key features of the active site surrounding the mononuclear iron that orient the substrate and provide a level of regiospecificity. ,,,− However, there is currently little insight into whether the various reactivities require different types of activated oxygen species or how such divergent species might evolve from a highly conserved metal center.…”