The active site of methanol dehydrogenase from methylobacterium extorquens
The active site of methanol dehydrogenase from methylobacterium extorquens
This thesis describes studies carried out on the active site of MDH purified from Methylobacterium extorquens on its interaction with cytochrome cL.
It has been previously proposed that Asp303 is the catalytic base necessary for methanol oxidation in MDH. In order to confirm its function, two site directed mutants of Asp303 were characterised. The aspartate residue was replaced by asparagine and glutamate (D303N and D303E) respectively. D303N-MDH was unable to grow on methanol as a sole carbon source and neither of the subunits of mutant MDH was syntehsized.D303E-MDH was able to grow on methanol and both subunits of mutant MDH were synthesized. The activity of pure D303E-MDH showed that the Km of this mutant of methanol and other substrates had increased from 3μM (for wild-type MDH) to 250mM. The Vmax for methanol oxidation in D303E-MDH had slightly increased to 1.2μmoles/min/mg from 0.8μmoles/min/mg for wild-type MDH. This resulted from a decrease in both activation energy and in the free energy for substrate binding in D303E-MDH. This suggested that catalysis in D303E-MDH involved an increase in the stabilisation of the transition state due to the construction of the substrate binding site by Glu303.Crystals of D303E-MDH were obtained that were suitable for structure determination at a resolution of 3Å. This structure confirmed that although the conformation of the active site of D303E-MDH was similar to WT-MDH, the carboxyl group of Glu303 interacted closely with the O5 of PQQ and the calcium ion. This confirmed the constriction of the substrate binding site, thereby resulting in the reduced affinity of D303E-MDH for methanol. This confirms the role of Asp303 as the catalytic base required for MDH activity.
University of Southampton
1999
Afolabi, Paul Remi
(1999)
The active site of methanol dehydrogenase from methylobacterium extorquens.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
This thesis describes studies carried out on the active site of MDH purified from Methylobacterium extorquens on its interaction with cytochrome cL.
It has been previously proposed that Asp303 is the catalytic base necessary for methanol oxidation in MDH. In order to confirm its function, two site directed mutants of Asp303 were characterised. The aspartate residue was replaced by asparagine and glutamate (D303N and D303E) respectively. D303N-MDH was unable to grow on methanol as a sole carbon source and neither of the subunits of mutant MDH was syntehsized.D303E-MDH was able to grow on methanol and both subunits of mutant MDH were synthesized. The activity of pure D303E-MDH showed that the Km of this mutant of methanol and other substrates had increased from 3μM (for wild-type MDH) to 250mM. The Vmax for methanol oxidation in D303E-MDH had slightly increased to 1.2μmoles/min/mg from 0.8μmoles/min/mg for wild-type MDH. This resulted from a decrease in both activation energy and in the free energy for substrate binding in D303E-MDH. This suggested that catalysis in D303E-MDH involved an increase in the stabilisation of the transition state due to the construction of the substrate binding site by Glu303.Crystals of D303E-MDH were obtained that were suitable for structure determination at a resolution of 3Å. This structure confirmed that although the conformation of the active site of D303E-MDH was similar to WT-MDH, the carboxyl group of Glu303 interacted closely with the O5 of PQQ and the calcium ion. This confirmed the constriction of the substrate binding site, thereby resulting in the reduced affinity of D303E-MDH for methanol. This confirms the role of Asp303 as the catalytic base required for MDH activity.
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Published date: 1999
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Local EPrints ID: 464223
URI: http://eprints.soton.ac.uk/id/eprint/464223
PURE UUID: 36088b10-48b6-4941-9270-8e510ff6613c
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Date deposited: 04 Jul 2022 21:37
Last modified: 04 Jul 2022 21:37
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Author:
Paul Remi Afolabi
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