Gentisate 1,2-dioxygenase from Pseudomonas. Substrate coordination to active site Fe2+ and mechanism of turnover

M. R. Harpel, J. D. Lipscomb

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Abstract

Gentisate 1,2-dioxygenase catalyzes the oxygenolytic ring cleavage of gentisate (2,5-dihydroxybenzoate) between carbons 1 and 2 to form maleylpyruvate. The essential active site Fe2+ of the enzyme binds NO to yield an EPR-active (S = 3/2) complex. Hyperfine broadening from 17O (I = 5/2) is observed in the spectrum of the enzyme-nitrosyl complex prepared in 17O-enriched water, demonstrating that water is an iron ligand. Association of gentisate with the enzyme-nitrosyl complex causes the broadening due to [17O]water to disappear, suggesting that water is displaced. Hyperfine broadening of the EPR spectrum for the gentisate bound complex is observed when 17O is incorporated into either the carbon 1 carboxylate or carbon 2 hydroxyl substituents of gentisate, but not when it is placed in the carbon 5 hydroxyl substituent. Thus, substrate apparently binds directly to the iron through the carbon 1 carboxylate and carbon 2 hydroxyl substituents, thereby bringing the site of ring cleavage close to the active site iron. Since NO must bind to the iron to elicit an EPR signal, a total of three sites in the iron coordination appear to be available for exogenous ligands. The role of the substrate functional groups in catalysis is investigated through comparison of the reaction kinetics of gentisate analogs using the gentisate 1,2-dioxygenases isolated from Pseudomonas acidovorans and Pseudomonas testosteroni. Turnover is either eliminated or substantially reduced on replacement of any of the functional groups of gentisate. Furthermore, an electron-donating frequency that can tautomerize (hydroxyl or amine) is required in a ring position either ortho or para to the carbon 2 substituent for turnover. The best alternate substrate of this group is 5-aminosalicylate, which is turned over at ~7% of the rate of gentisate by the enzyme from P. testosteroni. Both atoms from O2 are shown to be incorporated into the product of 5-aminosalicylate turnover. This is the first direct demonstration of dioxygenase stoichiometry in the reaction of any ferrous, non-heme, aromatic ring-cleaving dioxygenase. It is proposed that the enzyme-catalyzed O2 attack on the aromatic ring of gentisate is initiated from a complex in which O2 and substrate are simultaneously coordinated to the active site iron. Subsequent dioxygen bond cleavage and insertion are proposed to be promoted by a resonance shift involving ketonization of the carbon 5 hydroxyl group.

Original languageEnglish (US)
Pages (from-to)22187-22196
Number of pages10
JournalJournal of Biological Chemistry
Volume265
Issue number36
StatePublished - Dec 1 1990

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gentisate 1,2-dioxygenase
Gentisates
Pseudomonas
Catalytic Domain
Carbon
Hydroxyl Radical
Iron
Substrates
Comamonas testosteroni
Paramagnetic resonance
Enzymes
Dioxygenases
Mesalamine
Water
Functional groups
Delftia acidovorans
Ligands
Catalysis
Reaction kinetics
Stoichiometry

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Gentisate 1,2-dioxygenase from Pseudomonas. Substrate coordination to active site Fe2+ and mechanism of turnover. / Harpel, M. R.; Lipscomb, J. D.

In: Journal of Biological Chemistry, Vol. 265, No. 36, 01.12.1990, p. 22187-22196.

Research output: Contribution to journalArticle

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abstract = "Gentisate 1,2-dioxygenase catalyzes the oxygenolytic ring cleavage of gentisate (2,5-dihydroxybenzoate) between carbons 1 and 2 to form maleylpyruvate. The essential active site Fe2+ of the enzyme binds NO to yield an EPR-active (S = 3/2) complex. Hyperfine broadening from 17O (I = 5/2) is observed in the spectrum of the enzyme-nitrosyl complex prepared in 17O-enriched water, demonstrating that water is an iron ligand. Association of gentisate with the enzyme-nitrosyl complex causes the broadening due to [17O]water to disappear, suggesting that water is displaced. Hyperfine broadening of the EPR spectrum for the gentisate bound complex is observed when 17O is incorporated into either the carbon 1 carboxylate or carbon 2 hydroxyl substituents of gentisate, but not when it is placed in the carbon 5 hydroxyl substituent. Thus, substrate apparently binds directly to the iron through the carbon 1 carboxylate and carbon 2 hydroxyl substituents, thereby bringing the site of ring cleavage close to the active site iron. Since NO must bind to the iron to elicit an EPR signal, a total of three sites in the iron coordination appear to be available for exogenous ligands. The role of the substrate functional groups in catalysis is investigated through comparison of the reaction kinetics of gentisate analogs using the gentisate 1,2-dioxygenases isolated from Pseudomonas acidovorans and Pseudomonas testosteroni. Turnover is either eliminated or substantially reduced on replacement of any of the functional groups of gentisate. Furthermore, an electron-donating frequency that can tautomerize (hydroxyl or amine) is required in a ring position either ortho or para to the carbon 2 substituent for turnover. The best alternate substrate of this group is 5-aminosalicylate, which is turned over at ~7{\%} of the rate of gentisate by the enzyme from P. testosteroni. Both atoms from O2 are shown to be incorporated into the product of 5-aminosalicylate turnover. This is the first direct demonstration of dioxygenase stoichiometry in the reaction of any ferrous, non-heme, aromatic ring-cleaving dioxygenase. It is proposed that the enzyme-catalyzed O2 attack on the aromatic ring of gentisate is initiated from a complex in which O2 and substrate are simultaneously coordinated to the active site iron. Subsequent dioxygen bond cleavage and insertion are proposed to be promoted by a resonance shift involving ketonization of the carbon 5 hydroxyl group.",
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N2 - Gentisate 1,2-dioxygenase catalyzes the oxygenolytic ring cleavage of gentisate (2,5-dihydroxybenzoate) between carbons 1 and 2 to form maleylpyruvate. The essential active site Fe2+ of the enzyme binds NO to yield an EPR-active (S = 3/2) complex. Hyperfine broadening from 17O (I = 5/2) is observed in the spectrum of the enzyme-nitrosyl complex prepared in 17O-enriched water, demonstrating that water is an iron ligand. Association of gentisate with the enzyme-nitrosyl complex causes the broadening due to [17O]water to disappear, suggesting that water is displaced. Hyperfine broadening of the EPR spectrum for the gentisate bound complex is observed when 17O is incorporated into either the carbon 1 carboxylate or carbon 2 hydroxyl substituents of gentisate, but not when it is placed in the carbon 5 hydroxyl substituent. Thus, substrate apparently binds directly to the iron through the carbon 1 carboxylate and carbon 2 hydroxyl substituents, thereby bringing the site of ring cleavage close to the active site iron. Since NO must bind to the iron to elicit an EPR signal, a total of three sites in the iron coordination appear to be available for exogenous ligands. The role of the substrate functional groups in catalysis is investigated through comparison of the reaction kinetics of gentisate analogs using the gentisate 1,2-dioxygenases isolated from Pseudomonas acidovorans and Pseudomonas testosteroni. Turnover is either eliminated or substantially reduced on replacement of any of the functional groups of gentisate. Furthermore, an electron-donating frequency that can tautomerize (hydroxyl or amine) is required in a ring position either ortho or para to the carbon 2 substituent for turnover. The best alternate substrate of this group is 5-aminosalicylate, which is turned over at ~7% of the rate of gentisate by the enzyme from P. testosteroni. Both atoms from O2 are shown to be incorporated into the product of 5-aminosalicylate turnover. This is the first direct demonstration of dioxygenase stoichiometry in the reaction of any ferrous, non-heme, aromatic ring-cleaving dioxygenase. It is proposed that the enzyme-catalyzed O2 attack on the aromatic ring of gentisate is initiated from a complex in which O2 and substrate are simultaneously coordinated to the active site iron. Subsequent dioxygen bond cleavage and insertion are proposed to be promoted by a resonance shift involving ketonization of the carbon 5 hydroxyl group.

AB - Gentisate 1,2-dioxygenase catalyzes the oxygenolytic ring cleavage of gentisate (2,5-dihydroxybenzoate) between carbons 1 and 2 to form maleylpyruvate. The essential active site Fe2+ of the enzyme binds NO to yield an EPR-active (S = 3/2) complex. Hyperfine broadening from 17O (I = 5/2) is observed in the spectrum of the enzyme-nitrosyl complex prepared in 17O-enriched water, demonstrating that water is an iron ligand. Association of gentisate with the enzyme-nitrosyl complex causes the broadening due to [17O]water to disappear, suggesting that water is displaced. Hyperfine broadening of the EPR spectrum for the gentisate bound complex is observed when 17O is incorporated into either the carbon 1 carboxylate or carbon 2 hydroxyl substituents of gentisate, but not when it is placed in the carbon 5 hydroxyl substituent. Thus, substrate apparently binds directly to the iron through the carbon 1 carboxylate and carbon 2 hydroxyl substituents, thereby bringing the site of ring cleavage close to the active site iron. Since NO must bind to the iron to elicit an EPR signal, a total of three sites in the iron coordination appear to be available for exogenous ligands. The role of the substrate functional groups in catalysis is investigated through comparison of the reaction kinetics of gentisate analogs using the gentisate 1,2-dioxygenases isolated from Pseudomonas acidovorans and Pseudomonas testosteroni. Turnover is either eliminated or substantially reduced on replacement of any of the functional groups of gentisate. Furthermore, an electron-donating frequency that can tautomerize (hydroxyl or amine) is required in a ring position either ortho or para to the carbon 2 substituent for turnover. The best alternate substrate of this group is 5-aminosalicylate, which is turned over at ~7% of the rate of gentisate by the enzyme from P. testosteroni. Both atoms from O2 are shown to be incorporated into the product of 5-aminosalicylate turnover. This is the first direct demonstration of dioxygenase stoichiometry in the reaction of any ferrous, non-heme, aromatic ring-cleaving dioxygenase. It is proposed that the enzyme-catalyzed O2 attack on the aromatic ring of gentisate is initiated from a complex in which O2 and substrate are simultaneously coordinated to the active site iron. Subsequent dioxygen bond cleavage and insertion are proposed to be promoted by a resonance shift involving ketonization of the carbon 5 hydroxyl group.

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