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|Title:||Kinetic and structural studies of wild-type and recombinant aromatic amine dehydrogenase|
|Authors:||Abu-Khadra, Khalid Mohammad|
|Presented at:||University of Leicester|
|Abstract:||TTQ-dependent aromatic amine dehydrogenase (AADH) from Alcaligenes faecalis has been expressed heterologously in Paracoccus denitrificans via the cloning of the aromatic amine dehydrogenase utilizing genes (aau) under the regulation of the mauF promoter, another TTQ-dependent methylamine dehydrogenase (MADH). The recombinant AADH obtained during this study is completely active indicating complete biosynthesis of the TTQ cofactor by restricted cloning of only five genes (aauBEDA and orf-2) of the nine known genes which encode AADH. Chemical and physical characterization as well as kinetic analysis, show that the recombinant AADH expressed in Paracoccus denitrificans is indistinguishable from the native AADH expressed in Alcaligenes faecalis. The catalytic small subunit of AADH has been isolated and studied in the absence of the large subunit. Separation from the holoenzyme was achieved by chemical denaturation. The separation small subunit was refolded, characterized and studied using a number of different kinetic approaches. The small subunit shows a fully integrated and completely redox active TTQ centre. The ability of the separated small subunit to oxidase amine substrate was demonstrated, despite the very slow reaction rates when compared to the holoenzyme. The altered catalytic potency of the TTQ-containing small subunit revealed the significant role of the large subunit in providing the enzyme's catalytic power. Structural X-ray crystallographic studies were performed on crystalline enzyme in an attempt to elucidate further details about the reaction mechanism of enzyme and specifically the mode of C-H bond breakage. Structures of AADH in complex with different para substituted benzylamines were obtained. These structures provide valuable datasets for future studies using computational methods to address specific aspects of the chemistry of TTQ reduction by amine substrates.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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