The structure and function of methanol dehydrogenase
The structure and function of methanol dehydrogenase
This thesis describes studies carried out on the MDH in M.extroquens and its interaction with cytochrome cL.
Previous work had demonstrated that the interaction between MDH and cytochrome cL was ionic in nature and involved lysine residues on MDH forming interactions with carboxylate residues on cytochrome cL. In the present work, a program of chemical modification was undertaken in order to identify those lysines. Lysine residues on MDH were modified with TNBS and the modified MDH (TNP-MDH) was digested with thermolysin, peptides separated on reverse phase HPLC and the positions of the modified residues identified through peptide sequencing. Lysines 30, 205 and 583 were identified and proposed to be involved in interactions with cytochrome cL.
The chemical modification approach was discontinued in order to initiate a study of the active site by site directed mutagenesis. Three methods were attempted; the Kunkel method, the unique site elimination method and the QuikChangeTM method. Only the QuikChangeTM method was successful and is now used as the standard method of mutagenesis in this lab.
It had been previously proposed that a base was necessary in the active site of MDH in order to oxidise methanol. The position of Asp-303 in the X-ray structure made it the most likely candidate for the active site base. In order to confirm the function of Asp-303, site directed mutants were constructed in which the asparatate residue was replaced by glutamate and asparagine (D303E and D303N respectively). D33E was able to grow on methanol as sole carbon source. Activities in crude extracts showed that the Km of this mutant for methanol had increased from 20μM to 100mM while the Km for the activator (ammonia) was unchanged. D303N was difficult to interpret but was unable to grow on methanol as sole carbon source and could not be demonstrated to synthesize either of the MDH subunits.
University of Southampton
Majekodunmi, Enitan Oluwatosin
1997
Majekodunmi, Enitan Oluwatosin
Majekodunmi, Enitan Oluwatosin
(1997)
The structure and function of methanol dehydrogenase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
This thesis describes studies carried out on the MDH in M.extroquens and its interaction with cytochrome cL.
Previous work had demonstrated that the interaction between MDH and cytochrome cL was ionic in nature and involved lysine residues on MDH forming interactions with carboxylate residues on cytochrome cL. In the present work, a program of chemical modification was undertaken in order to identify those lysines. Lysine residues on MDH were modified with TNBS and the modified MDH (TNP-MDH) was digested with thermolysin, peptides separated on reverse phase HPLC and the positions of the modified residues identified through peptide sequencing. Lysines 30, 205 and 583 were identified and proposed to be involved in interactions with cytochrome cL.
The chemical modification approach was discontinued in order to initiate a study of the active site by site directed mutagenesis. Three methods were attempted; the Kunkel method, the unique site elimination method and the QuikChangeTM method. Only the QuikChangeTM method was successful and is now used as the standard method of mutagenesis in this lab.
It had been previously proposed that a base was necessary in the active site of MDH in order to oxidise methanol. The position of Asp-303 in the X-ray structure made it the most likely candidate for the active site base. In order to confirm the function of Asp-303, site directed mutants were constructed in which the asparatate residue was replaced by glutamate and asparagine (D303E and D303N respectively). D33E was able to grow on methanol as sole carbon source. Activities in crude extracts showed that the Km of this mutant for methanol had increased from 20μM to 100mM while the Km for the activator (ammonia) was unchanged. D303N was difficult to interpret but was unable to grow on methanol as sole carbon source and could not be demonstrated to synthesize either of the MDH subunits.
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Published date: 1997
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Local EPrints ID: 462987
URI: http://eprints.soton.ac.uk/id/eprint/462987
PURE UUID: ab749f83-30d7-4996-bf5f-3a3c6bea28d9
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Date deposited: 04 Jul 2022 20:34
Last modified: 04 Jul 2022 20:34
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Author:
Enitan Oluwatosin Majekodunmi
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