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Structural studies of cytochrome P450 BM3 and CPRK

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posted on 15.12.2014, 10:32 by Michael Gordon Joyce
This work presents the crystal structure determination of the transcriptional regulator CprK and of individual domains of the multidomain cytochrome P450-BM3. The crystal structure of the A264E mutant heme domain was determined with and without substrate present. Surprisingly, the structures reveal the protein to exhibit a substrate bound conformation regardless of the presence of substrate. This has provided further evidence that substrate binding leads to a dramatic shift in the equilibrium of conformational states available to the protein. In addition, the crystal structure of the C773A mutant flavin binding domain has been determined both in presence and absence of NADP+. Together with the already available structures of the other domains, this now allows both modelling and further solution studies of the full length cytochrome P450-BM3 structure.;Dehalogenans sp. are capable of using a range of chlorophenolic compounds as terminal electron acceptors in a respiratory metabolism known as halorespiration. This process is under transcriptional control by CprK, a member of the CRP-FNR family of transcriptional regulators. The crystal structure of D. hafniense CprK in complex with o-chlorophenolacetic acid (OCPA) reveals tightly bound effector molecules. Binding of OCPA is analysed through both mutagenesis and fluorescence quenching binding studies. The results have led to the hypothesis that CprK uses the bound phenolic compound pKa as an additional mechanism to sense the presence of the chloride atom. The structure presents a structural framework for further studies of the mechanism of this family of transcriptional regulators and of CprK homologues in particular.

History

Date of award

01/01/2005

Author affiliation

Biochemistry

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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