Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.642144
Title: Structural studies on heme containing proteins
Author: Bruckmann, Chiara
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
Availability of Full Text:
Access through EThOS:
Full text unavailable from EThOS. Please try the link below.
Access through Institution:
Abstract:
Thus thesis reports the high-resolution structures of four different heme-containing enzymes: two dioxygenases (tryptophan 2,3-dioxygenase and an indoleamine 2,3-dioxygenase), and mutant forms of flavocytochrome b2 and nitric oxide synthase. We have solved the x-ray crystal structure of TDO from Xanthomonas campestris in the apo-form (at 2.7 Å resolution) and with ferric heme at the active site (at 2.6 Å resolution). Two 1.6 Å and 1.8 Å resolution structures of the catalytically active, ferrous form of the enzyme in binary complex (respectively with the substrate L-tryprophan and with the substrate analogue 6-fluoro-tryptophan) have also been determined. These data allow important insights into substrate recognition, defining the substrate specificity. The structure shows that the enzyme is a tetramer with a heme cofactor and a substrate molecule bound at each active site. A second, possibly allosteric, L-Trip-binding site is also identified at the tetramer interface. The active site is fully formed only in the binary complex, showing that TDO is an induced-fit enzyme with significant structural changes on the binding of substrate. The structure of TDO in complex with L-Trp revealed that histidine 55 hydrogen bonds to substrate, helping to correctly position it in the active site and possibly being implicated as a catalytic base during the reaction. Histidine 55 was replaced by alanine (H55A) by site-directed mutagenesis. The crystal structure of TDO H55A has been solved to 2.15 Å resolution in a new crystal form, in the presence of the substrate L-Trp. The different crystal form stabilizes the N-terminal region of the H55A mutant enzyme, which is better defined than in wild-type TDO.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.642144  DOI: Not available
Share: