Studies on enzymes of the 3-phenylpropionate catabolic pathway from Escherichia coli
Studies on enzymes of the 3-phenylpropionate catabolic pathway from Escherichia coli
The 3-phenylpropionate catabolic pathway found in Escherichia coli is a member of the family of oxidative meta-cleavage pathways employed by bacteria in the degradation of aromatic molecules. The enzyme 2-hydroxy penta-2,4-dienoic acid hydratase (MhpD) catalyses the hydration of 2-hydroxy penta-2,4-dienoic acid (16) to afford 4-hydroxy-2-keto pentanoic acid (21). (21) is then cleaved in a retro-aldol reaction catalysed by 4-hydroxy-2-keto pentanoic acid aldolase (MhpE), to yield pyruvic acid and acetaldehyde. This thesis describes the studies toward the purification and characterisation of the enzymes MhpD and MhpE and the characterisation of the two reactions catalysed.
(16) was isolated by the conversion of 2-hydroxy-6-oxo-nona-2,4-diene-1,9-dioic acid (15) using partially purified MhpC and its physical and chemical properties characterised.
MhpD was purified 3700 fold to homogeneity from an overexpressing strain of Escherichia coli. The purified 28 kDa protein required a divalent metal ion for activity, with optimum activity being obtained with Mn2+. A KM of 142 μM and a kcat of 1580 s-1 were measured for the substrate (16). The mechanism of the MhpD catalysed reaction was probed using analogues of possible reaction intermediates as potential inhibitors. Sodium oxalate was found to act as a potent inhibitor with a K1 of 4.9 μM. Inhibition by oxalate is consistent with a mechanism in which tautomerisation to 2-keto pent-3-enoic acid (18) takes place at the enzyme active site, followed by conjugate addition of water to afford an enolate intermediate. A route to determine the absolute stereochemistry at C-4 was established, which involved the three step derivatisation of (21) to afford a diastereotopic product which was resolved by 1H and 19F NMR spectroscopy. Stereochemistry at C-5 was studied using a chiral methyl strategy. (21) containing a chiral methyl group at C-5 was prepared by the reaction of MhpD on [5E-2H, 5z-3H]-(15) in H2O and [5z-3H]-(15) in D2O. These were then oxidised to chiral acetic acid in preparation for chiral analysis.
MhpE was partially purified (28 fold) from wild type E. coli. The enzyme was completely inhibited by NaBH4 in the presence of (21) implying the mechanism operates via a Schiff base intermediate, allowing the classification of MhpE as a type II aldolase. MhpE consumed up to 50% of a racemic pool of (21) indicating the stereospecificity of the enzyme, contradicting early studies of the enzyme in cell free extracts.
University of Southampton
1997
Pollard, John Robert
(1997)
Studies on enzymes of the 3-phenylpropionate catabolic pathway from Escherichia coli.
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 in the degradation of aromatic molecules. The enzyme 2-hydroxy penta-2,4-dienoic acid hydratase (MhpD) catalyses the hydration of 2-hydroxy penta-2,4-dienoic acid (16) to afford 4-hydroxy-2-keto pentanoic acid (21). (21) is then cleaved in a retro-aldol reaction catalysed by 4-hydroxy-2-keto pentanoic acid aldolase (MhpE), to yield pyruvic acid and acetaldehyde. This thesis describes the studies toward the purification and characterisation of the enzymes MhpD and MhpE and the characterisation of the two reactions catalysed.
(16) was isolated by the conversion of 2-hydroxy-6-oxo-nona-2,4-diene-1,9-dioic acid (15) using partially purified MhpC and its physical and chemical properties characterised.
MhpD was purified 3700 fold to homogeneity from an overexpressing strain of Escherichia coli. The purified 28 kDa protein required a divalent metal ion for activity, with optimum activity being obtained with Mn2+. A KM of 142 μM and a kcat of 1580 s-1 were measured for the substrate (16). The mechanism of the MhpD catalysed reaction was probed using analogues of possible reaction intermediates as potential inhibitors. Sodium oxalate was found to act as a potent inhibitor with a K1 of 4.9 μM. Inhibition by oxalate is consistent with a mechanism in which tautomerisation to 2-keto pent-3-enoic acid (18) takes place at the enzyme active site, followed by conjugate addition of water to afford an enolate intermediate. A route to determine the absolute stereochemistry at C-4 was established, which involved the three step derivatisation of (21) to afford a diastereotopic product which was resolved by 1H and 19F NMR spectroscopy. Stereochemistry at C-5 was studied using a chiral methyl strategy. (21) containing a chiral methyl group at C-5 was prepared by the reaction of MhpD on [5E-2H, 5z-3H]-(15) in H2O and [5z-3H]-(15) in D2O. These were then oxidised to chiral acetic acid in preparation for chiral analysis.
MhpE was partially purified (28 fold) from wild type E. coli. The enzyme was completely inhibited by NaBH4 in the presence of (21) implying the mechanism operates via a Schiff base intermediate, allowing the classification of MhpE as a type II aldolase. MhpE consumed up to 50% of a racemic pool of (21) indicating the stereospecificity of the enzyme, contradicting early studies of the enzyme in cell free extracts.
This record has no associated files available for download.
More information
Published date: 1997
Identifiers
Local EPrints ID: 463070
URI: http://eprints.soton.ac.uk/id/eprint/463070
PURE UUID: 9c977b3e-62fd-4c53-a93e-e9a060beb9f3
Catalogue record
Date deposited: 04 Jul 2022 20:43
Last modified: 04 Jul 2022 20:43
Export record
Contributors
Author:
John Robert Pollard
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics