Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

Eppinger, Erik; Ferraroni, Marta; Bürger, Sibylle; Steimer, Lenz; Peng, Grace C.Y.; Briganti, Fabrizio

doi:10.1016/j.bbapap.2015.06.005

Cited by 14 publications

(52 citation statements)

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“…The three His ligandsi nS DO are arranged on one face of the octahedral metal coordination geometry, while other sites are availablef or the substrate and O 2 .I nterestingly, SDO is the first enzyme discovered to catalyze the ring fission of am onohydroxylated aromatic compound, salicylate. [13] O 2 attacks the enzyme-substrate complex (E)t of orm aF e-O 2 adduct, whichf orms an alkylperoxo-bridged compound (AP). [12c] Thus, the substrates in other dioxygenases are activated by virtue of dihydroxyc ompounds or contain at least one electron-donating group in the aromatic ring.…”

Section: Introductionmentioning

confidence: 99%

“…[12c] Thus, the substrates in other dioxygenases are activated by virtue of dihydroxyc ompounds or contain at least one electron-donating group in the aromatic ring. [13] We are not aware of any computational studies exploring the reactionm echanism of SDO as well as GDO in general, whereas there is ar ich literature for both class Ia nd II (i.e. Moreover, SDO is av ersatile enzymec apable of catalyzingt he cleavage of aw ide range of substrates rangingf rom salicylate to gentisate to 1-hydroxy-2-napthaoate.I ntra-diol and extra-diol enzymesa re generally categorized as class Ia nd II dioxygenases, respectively,w hile SDO has been assigned as ac lass III dioxygenasei nc onjunction with gentisate 1,2-dioxygenase (GDO).…”

Section: Introductionmentioning

confidence: 99%

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Catalytic Mechanism of Salicylate Dioxygenase: QM/MM Simulations Reveal the Origin of Unexpected Regioselectivity of the Ring Cleavage

Roy

Kästner

2017

Chemistry A European J

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Salicylate 1,2-dioxygenase (SDO) was the first enzyme discovered, in the family of iron dioxygenases, to catalyze the ring cleavage of a monohydroxylated aromatic compound, salicylate, without a proton donor. Salicylate is not electron-rich like the familiar dihydroxy or aromatic substrates with an electron-donating group that are utilized in the well-known dioxygenases. SDO carries out the intramolecular C-C bond cleavage in salicylate bearing the OH and COOH groups with high regioselectivity in comparison with the extradiol and intradiol dioxygenases. The catalytic cleavage of a nonactivated substrate like salicylate that lacks an electron-donating group, also in the absence of a proton source, raises many puzzling questions about the oxy intermediates in the reaction pathway of dioxygenase enzymes in general. To answer these fundamental queries, we have investigated the full catalytic mechanism of SDO by a combination of quantum mechanics and molecular mechanics (QM/MM) calculations. Herein, our QM/MM study has several unexpected and interesting implications for the mechanistic pathway of SDO in comparison to the experimental observations. Importantly, it unravels the basis for the unexpected "intra"-cleavage regioselectivity in SDO. Ostensibly a similar alkylperoxo intermediate is formed in SDO much like in the extradiol and intradiol dioxygenases. In stark contrast to the two diol enzymes, the O-O bond breaking leads to an unprotonated gem-hydroxy carboxylate intermediate, a paradigm analogue of the elusive gem diol intermediate. This unprotonated gem-hydroxy carboxylate intermediate exclusively dictates the C-C cleavage regiospecificity in SDO, which is unprecedented in the family of dioxygenases. It forms a seven-membered lactone species, which eventually forms the ring-cleavage final product by incorporation of two oxygen atoms in the salicylate. Thus, our computational study unravels a detailed reaction pathway of the oxidative cleavage of salicylate without a proton source by identifying the hitherto elusive intermediates in the catalytic cycle of SDO with testable predictions. Moreover, we describe the atomistic origin of the new catalytic role of Arg127 in the catalysis and regioselectivity of SDO. It also rationalizes the formation of a side product without invoking a dioxetane intermediate. This study is important from a fundamental perspective and opens up a new window in the mechanism of the family of dioxygenase enzymes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Catalytic Mechanism of Salicylate Dioxygenase: QM/MM Simulations Reveal the Origin of Unexpected Regioselectivity of the Ring Cleavage

Roy

Kästner

2017

Chemistry A European J

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“…Mutation experiments 15 have shown that His162 is essential for catalysis, because the His162Ala mutant was almost inactive. However, when Arg127 was mutated to Ile, Asn, or Lys, no significant loss of activity was observed.…”

Section: Exploring the Roles Of Arg127 And His162mentioning

confidence: 99%

“…In SDO, residues Arg127 and His162 may play a similar role. 5 Mutation studies 14,15 have shown that His162 is essential for catalysis: The His162Ala SDO mutant lost almost all activities when reacting with salicylate. However, when Arg127 was mutated to Ile, Asn, or Lys, no significant loss of activity was observed.…”

Section: Scheme 1 Overall Reaction Catalysed By Sdomentioning

confidence: 99%

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O₂ Activation in Salicylate 1,2-Dioxygenase: A QM/MM Study Reveals the Role of His162

Dong

Ryde

2016

Inorg. Chem.

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QM/MM‐Simulationen ergeben synergetische Substrat‐ und Sauerstoffaktivierung in Salicylat‐Dioxygenase

Roy

Kästner

2015

Angewandte Chemie

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Salicylat‐1,2‐Dioxygenase (SDO) ist das erste bekannte Enzym, das die oxidative Spaltung eines einfach hydroxylierten Aromaten, Salicylat, statt der wohlbekannten elektronenreichen Substrate katalysiert. Wir haben den Mechanismus der Sauerstoffaktivierung in SDO mit QM/MM‐Simulationen untersucht. Unsere Ergebnisse zeigen, dass das Nicht‐Häm‐FeII‐Zentrum in SDO Salicylat und O2 gemeinsam durch eine starke kovalente Wechselwirkung aktiviert, um die reduktive Spaltung von O2 zu ermöglichen. Die reaktive Sauerstoffspezies ist ein kovalenter Salicylat‐FeII‐O2‐Komplex, wobei die Elektronenstruktur zwischen den beiden Grenzfällen FeII‐O2 und FeII‐O2.− mit partiellem Elektronentransfer vom aktivierten Salicylat zu O2 über das Fe‐Zentrum liegt. Folglich verwendet SDO eine synergetische Strategie der Substrat‐ und Sauerstoffaktivierung, um die katalytische Reaktion auszuführen, die einmalig in der Familie der Eisendioxygenasen ist. Darüber hinaus erfolgt die O2‐Aktivierung in SDO ohne die Hilfe einer Protonenquelle. Unsere Studie zeigt eine bisher unbekannte Möglichkeit für den Mechanismus der O2‐Aktivierung.

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Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

Cited by 14 publications

References 50 publications

Catalytic Mechanism of Salicylate Dioxygenase: QM/MM Simulations Reveal the Origin of Unexpected Regioselectivity of the Ring Cleavage

Catalytic Mechanism of Salicylate Dioxygenase: QM/MM Simulations Reveal the Origin of Unexpected Regioselectivity of the Ring Cleavage

O₂ Activation in Salicylate 1,2-Dioxygenase: A QM/MM Study Reveals the Role of His162

QM/MM‐Simulationen ergeben synergetische Substrat‐ und Sauerstoffaktivierung in Salicylat‐Dioxygenase

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Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

Cited by 14 publications

References 50 publications

Catalytic Mechanism of Salicylate Dioxygenase: QM/MM Simulations Reveal the Origin of Unexpected Regioselectivity of the Ring Cleavage

Catalytic Mechanism of Salicylate Dioxygenase: QM/MM Simulations Reveal the Origin of Unexpected Regioselectivity of the Ring Cleavage

O2 Activation in Salicylate 1,2-Dioxygenase: A QM/MM Study Reveals the Role of His162

QM/MM‐Simulationen ergeben synergetische Substrat‐ und Sauerstoffaktivierung in Salicylat‐Dioxygenase

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O₂ Activation in Salicylate 1,2-Dioxygenase: A QM/MM Study Reveals the Role of His162