Design principles for site-selective hydroxylation by a Rieske oxygenase

Liu, Jianxin; Tian, Jiayi; Perry, Christopher; Lukowski, April L.; Doukov, Tzanko I.; Narayan, Alison R. H.; Bridwell-Rabb, Jennifer

doi:10.1038/s41467-021-27822-3

Title: Design principles for site-selective hydroxylation by a Rieske oxygenase

Journal Article · 11 January 2022 · Nature Communications

DOI: https://doi.org/10.1038/s41467-021-27822-3 · OSTI ID:1862316

^[1]; Tian, Jiayi ^[1]; Perry, Christopher ^[1]; Lukowski, April L. ^[1];

^[2];

^[1];

^[1]

Univ. of Michigan, Ann Arbor, MI (United States)
SLAC National Accelerator Lab., Menlo Park, CA (United States)

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.

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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); Rackham Graduate School (UM); National Institute of General Medical Sciences

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1862316

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 13; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Title: Design principles for site-selective hydroxylation by a Rieske oxygenase

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