Evolutionary adaptation from hydrolytic to oxygenolytic catalysis

Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading N-heteroaromatic substrates belong to the /{beta}-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. Here, we have integrated crystallographic analyses at normoxic and hyperoxic conditions with molecular dynamics and quantum mechanical calculations to investigate its prototypic 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) member. O2 localization to the "oxyanion hole", where catalysis occurs, is an unfavorable event and the direct competition between dioxygen and water for this site is modulated by the "nucleophilic elbow" residue. A hydrophobic pocket that overlaps with the organic substrate binding site can act as a proximal dioxygen reservoir. Freeze-trap pressurization allowed to determine the structure of the ternary complex with a substrate analogue and O2 bound at the oxyanion hole. Theoretical calculations reveal that O2 orientation is coupled to the charge of the bound organic ligand. When 1-H-3-hydroxy-4-oxoquinaldine is uncharged, O2 binds with its molecular axis along the ligands C2-C4 direction in full agreement with the crystal structure. Substrate activation triggered by deprotonation of its 3-OH group by the His-Asp dyad, rotates O2 by approximately 60 degrees. This geometry maximizes the charge-transfer between the substrate and O2 thus weakening the double bond of the latter. Electron density transfer to the O2({pi}*) orbital promotes the formation of the peroxide intermediate via intersystem crossing that is rate-determining. Our work provides a detailed picture of how evolution has repurposed the ABH-fold architecture and its simple catalytic machinery to accomplish metal-independent oxygenation. SignificanceMany of the current O2-dependent enzymes have evolved from classes that existed prior to the switch from a reducing to an oxidative atmosphere and whose original functions are unrelated to dioxygen chemistry. A group of bacterial dioxygenases belong to the /{beta}-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. These enzymes degrade their N-heteroaromatic substrates in a cofactor-independent manner relying only on the simple nucleophile-histidine-acid ABH-fold catalytic toolbox. In this work we show how O2 localizes at the catalytic site by taking advantage of multiple strategies that minimize the strong competition by water, the co-substrate in the ancestral hydrolytic enzyme. We also show that substrate activation by the His-Asp catalytic dyad leads a ligand-O2 complex that maximizes the electron transfer from the organic substrate to O2, thus promoting intersystem crossing and circumventing the spin-forbiddeness of the reaction. Overall, our work explains how evolution has repurposed the ABH-fold architecture and its simple catalytic machinery to accomplish spin-restricted metal-independent oxygenation.