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Characterization of the 4-carboxy-2-hydroxymuconate hydratase and the 4-carboxy-4-hydroxy-2-oxoadipate/4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas putida

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dc.contributor.advisor Seah, Stephen
dc.contributor.author Mazurkewich, Scott
dc.date.accessioned 2016-05-11T20:05:13Z
dc.date.available 2017-05-01T05:00:13Z
dc.date.copyright 2016-05
dc.date.created 2016-05-04
dc.date.issued 2016-05-11
dc.identifier.uri http://hdl.handle.net/10214/9677
dc.description.abstract The protocatechuate and gallate 4,5-cleavage pathways are important bacterial catabolic pathways for environmental carbon cycling and xenobiotic remediation. This thesis focuses on the characterization of the 4-carboxy-2-hydroxymuconate (CHM) hydratase and the 4-hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase which are the last two steps of the protocatechuate and gallate 4,5-cleavage pathways. HMG/CHA aldolases are class II pyruvate aldolases that have structural similarity to a group of proteins termed Regulators of RNase E activity A (RraA). The Escherichia coli RraA (EcRraA) binds to the regulatory domain of RNase E, inhibiting ribonuclease activity. Sequence and kinetic analyses of homologous RraA-like proteins identified minimal motifs, either a D-X20-R-D or a G-X20-R-D-X2-E/D motif, required for metal binding and aldolase activity amongst homologs. The EcRraA, which lacked sequence conservation to either motif, lacked detectable C-C lyase activity. Upon restoration of the G-X20-R-D-X2-E/D motif, the EcRraA was able to catalyze oxaloacetate decarboxylation. RraA-like gene products are found across all the domains of life with a large proportion containing one of the sequence motifs, implying that the proteins likely support a metal dependent enzyme function. The HMG/CHA aldolase is activated in the presence of inorganic phosphate (Pi), increasing its turnover rate >10-fold, which is unique for a class II aldolase. The HMG/CHA aldolases pyruvate methyl proton exchange rate was increased 300-fold in the presence of 1 mM Pi. Docking studies revealed a potential Pi binding site close to the proposed general acid water site. The residues which comprise the putative binding pocket were probed through mutagenesis studies with substitution of either of the residues leading to a reduction in Pi activation, supporting the mechanism of Pi activation through the general acid half reaction. The CHM hydratase (GalB) from the gallate degradation pathway of Pseudomonas putida KT2440 has only 12% sequence identity to a previously identified CHM hydratase (LigJ) from Sphingomonas sp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-L N-acetyl glucosamine deacetylase family, and is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereo-specificity as GalB, providing an example of convergent evolution in bacterial aromatic degradation pathways. en_US
dc.language.iso en en_US
dc.subject aldolase en_US
dc.subject metalloenzyme en_US
dc.subject 4-carboxy-2-hydroxymuconate en_US
dc.subject 4-carboxy-4-hydroxy-2-oxoadipate en_US
dc.subject hydratase en_US
dc.subject Pseudomonas en_US
dc.subject lignin en_US
dc.subject 4-oxalomesaconate en_US
dc.subject protocatechuate en_US
dc.subject gallate en_US
dc.subject RraA en_US
dc.title Characterization of the 4-carboxy-2-hydroxymuconate hydratase and the 4-carboxy-4-hydroxy-2-oxoadipate/4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas putida en_US
dc.type Thesis en_US
dc.degree.programme Molecular and Cellular Biology en_US
dc.degree.name Doctor of Philosophy en_US
dc.degree.department Department of Molecular and Cellular Biology en_US
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