Studies an extradiol catechol dioxygenases

Spence, Emma Louise (1996) Studies an extradiol catechol dioxygenases. University of Southampton, Doctoral Thesis.

Abstract

The 3-phenyl propionate pathway found in Escherichia coli is one of a family of extradiol cleavage pathways which are used for bacterial degradation of aromatic compounds. The enzymes 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) and 2-hydroxy-6-oxo-nona-2,4-diene-1,9-dionate 5,6-hydrolase (MhpC) catalyse the third and fourth steps of the pathway. MhpB catalyses the extradiol cleavage of 2,3-dihydroxyphenylpropionate acid (8) to give 2-hydroxy-6-oxo-nona-2,4-diene-1,9-dionate (9). (9) is then cleaved by the hydrolase MhpC to give succinate and 2-hydroxypenta-2,4-dienoate (10).

MpcI from Alcaligenes eutrophus is another catechol extradiol dioxygenase. This thesis describes the purification of MpcI; a comparison of the properties of MhpB and MpcI and studies of the mechanism of extradiol catechol dioxygenases.

Substrate analogues cis- and trans-2-(2,3-dihydroxyphenyl)cyclopropane-1-carboxylic acid 39a) and (39b) were synthesised as probes for a semiquinone radical intermediate in the MhpB reaction. These analogues were found to be substrates for MhpB and MpcI. The stereochemistry of the ring fission products (41z) and (49b) was analysed by conversion to cyclopropane-1,2-dicarboxylic acids using MhpC, followed by GCMS analysis. This analysis revealed 85-94% trans-product and 6-15 % cis-product, implying that cis/trans isomerisation of the cyclopropyl ring substituents had taken place during the enzymatic conversion. These results are consistent with a reversible opening of the cyclopropyl ring, and hence consistent with the intermediacy of a semiquinone radical intermediate in the extradiol catechol dioxygenase reaction.

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