Crystal structure of the cofactor-devoid 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) catalytically inactive H251A variant complexed with 2-methyl-quinolin-4(1H)-one under normoxic conditions
Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading N-heteroaromatic substrates belong to the alpha/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.
Significance: Many 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 alpha/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.
Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading N-heteroaromatic substrates belong to the alpha/beta-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. Here, we have integrated crystallographic analyses under 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 the structure of the ternary complex with a substrate analogue and O2 bound at the oxyanion hole to be determined. 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 ligand's C2C4 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 deg. 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***LongTextEnd***
Paper "Ahmadi-Soleimani_2023_Life.Sci__122100"
Author "Ahmadi-Soleimani SM"
Author "Amiry GY"
Author "Khordad E"
Author "Masoudi M"
Author "Beheshti F"
Year "2023"
Title "Omega-3 fatty acids prevent nicotine withdrawal-induced impairment of learning and memory via affecting oxidative status, inflammatory response, cholinergic activity, BDNF and amyloid-B in rat hippocampal tissues"
Journal "Life Sciences"
Volume ""
Page "122100" ""
Medline "37722588"
Abstract "Ahmadi-Soleimani_2023_Life.Sci__122100"
LongText "Ahmadi-Soleimani_2023_Life.Sci__122100"
In the present study, the main objective was to reveal whether treatment by Omega-3 fatty acids could prevent the adverse effects of adolescent nicotine withdrawal on spatial and avoidance memory in male rats. For this purpose, Morris water maze and passive avoidance tests were performed on male Wistar rats and the hippocampal levels of oxidative stress markers, inflammatory indices, brain-derived neurotrophic factor, nitrite, amyloid-B and acetylcholinesterase (AChE) were measured. Moreover, density of dark neurons were assessed in CA(1) and CA(3) regions. Results showed that adolescent nicotine exposure followed by a period of drug cessation exacerbates the behavioral indices of learning and memory through affecting a variety of biochemical markers within the hippocampal tissues. These changes lead to elevation of oxidative and inflammatory markers, reduction of neurotrophic capacity and increased AChE activity in hippocampal tissues. In addition, it was observed that co-administration of nicotine with Omega-3 fatty acids significantly prevents nicotine withdrawal-induced adverse effects through restoration of the mentioned biochemical disturbances. Therefore, we suggest administration of Omega-3 fatty acids as a safe, inexpensive and effective therapeutic strategy for prevention of memory dysfunctions associated with nicotine abstinence during adolescence.
        
Representative scheme of HOD-cofactorfree-dioxygenase structure and an image from PDBsum server
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7OJM Previously Class, Architecture, Topology and Homologous superfamily - PDB-Sum server
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7OJMFold classification based on Structure-Structure alignment of Proteins - FSSP server