Mechanistic studies on 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase
Mechanistic studies on 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase
The 3-phenylpropionate catabolic pathway found in Escherichia coli is a member of the family of oxidative meta-cleavage pathways employed by bacteria for the degradation of aromatic molecules. The enzymes 2,3-dihydroxyphenyl-propionate 1,2-dioxygenase (MhpB) and 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioate 5,6-hydrolase (MhpC) catalyse the third and fourth steps respectively of the pathway. MhpB catalyses the meta-ring cleavage reaction of 2,3-dihydroxyphenyl-propionate (38) to give 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioate (39). (39) is then hydrolytically cleaved by MhpC to give succinate and 2-hydroxy-penta-2,4-dienoate (41). This thesis describes studies on the mechanism of the reaction catalysed by MhpC and the development of a synthetic route towards the dienol substrates of MhpC.
Observation of 4.5% incorporation of two atoms of 18O into succinate (40) from the MhpC catalysed cleavage of (39) in 95% H218O is consistent with a gem-diol intermediate suggesting that the reaction catalysed by MhpC proceeds via a general base mechanism. Isolation of 2-keto-6-hydroxy-octa-3-enoate, ethyl ester (62) and 2,6-dihydroxy-octa-2,4-dienoate, ethyl ester (63) from the reverse reaction catalysed by MhpC of propionaldehyde with 2-keto-penta-4-enoate, ethyl ester (61) is also consistent with the reaction catalysed by MhpC proceeding via a general base mechanism.
Studies on the non-enzyme base catalysed cleavage of (57) by the identification of the production of 14C-propionic acid have shown a time dependent reaction. Treatment of (39) with Na18OH has shown incorporation of two atoms of 18O which is consistent with the reversible formation of a tetrahedral intermediate in the reaction. These two experiments provide chemical precedent for a base catalysed reaction and supporting evidence for MhpC catalysing the reaction via a general base mechanism.
Two synthetic routes towards the dienol substrates of MhpC have been investigated. γ-Alkylation of trimethylsiloxy-penta-2,4-dienoic acid, ethyl ester (90) with various acetals of propionaldehyde in the presence of a catalytic amount of a Lewis acid has led to the synthesis of 2-keto-6-ethoxy-octa-3-enoic acid, ethyl ester (92) and 2-keto-6-allyloxy-octa-3-enoic acid, ethyl ester (99). Thus, it has been shown that the carbon skeleton for the dienol substrates of MhpC can be reproducibly synthesised, containing all the required functional groups.
University of Southampton
1997
Fleming, Sarah Margaret
(1997)
Mechanistic studies on 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The 3-phenylpropionate catabolic pathway found in Escherichia coli is a member of the family of oxidative meta-cleavage pathways employed by bacteria for the degradation of aromatic molecules. The enzymes 2,3-dihydroxyphenyl-propionate 1,2-dioxygenase (MhpB) and 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioate 5,6-hydrolase (MhpC) catalyse the third and fourth steps respectively of the pathway. MhpB catalyses the meta-ring cleavage reaction of 2,3-dihydroxyphenyl-propionate (38) to give 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioate (39). (39) is then hydrolytically cleaved by MhpC to give succinate and 2-hydroxy-penta-2,4-dienoate (41). This thesis describes studies on the mechanism of the reaction catalysed by MhpC and the development of a synthetic route towards the dienol substrates of MhpC.
Observation of 4.5% incorporation of two atoms of 18O into succinate (40) from the MhpC catalysed cleavage of (39) in 95% H218O is consistent with a gem-diol intermediate suggesting that the reaction catalysed by MhpC proceeds via a general base mechanism. Isolation of 2-keto-6-hydroxy-octa-3-enoate, ethyl ester (62) and 2,6-dihydroxy-octa-2,4-dienoate, ethyl ester (63) from the reverse reaction catalysed by MhpC of propionaldehyde with 2-keto-penta-4-enoate, ethyl ester (61) is also consistent with the reaction catalysed by MhpC proceeding via a general base mechanism.
Studies on the non-enzyme base catalysed cleavage of (57) by the identification of the production of 14C-propionic acid have shown a time dependent reaction. Treatment of (39) with Na18OH has shown incorporation of two atoms of 18O which is consistent with the reversible formation of a tetrahedral intermediate in the reaction. These two experiments provide chemical precedent for a base catalysed reaction and supporting evidence for MhpC catalysing the reaction via a general base mechanism.
Two synthetic routes towards the dienol substrates of MhpC have been investigated. γ-Alkylation of trimethylsiloxy-penta-2,4-dienoic acid, ethyl ester (90) with various acetals of propionaldehyde in the presence of a catalytic amount of a Lewis acid has led to the synthesis of 2-keto-6-ethoxy-octa-3-enoic acid, ethyl ester (92) and 2-keto-6-allyloxy-octa-3-enoic acid, ethyl ester (99). Thus, it has been shown that the carbon skeleton for the dienol substrates of MhpC can be reproducibly synthesised, containing all the required functional groups.
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Published date: 1997
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Local EPrints ID: 463120
URI: http://eprints.soton.ac.uk/id/eprint/463120
PURE UUID: f5686f7d-8c9f-4499-bfe1-9d95eaba148d
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Date deposited: 04 Jul 2022 20:45
Last modified: 04 Jul 2022 20:45
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Author:
Sarah Margaret Fleming
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