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Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L)

Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L)
Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L)
Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 Å resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome cL, which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.
0006-2960
9799-9809
Afolabi, P.R.
757e7f01-664c-493e-bc51-c6a2c933dc22
Mohammed, F.
e6dd5a98-bea5-484a-873a-da499c034594
Amaratunga, K.
6b6df5e8-b731-4c34-b1b8-8fc765fa1459
Majekodunmi, O.
c6df2337-0556-48e7-b390-b3d040d501d9
Dales, S.L.
a2c11795-0632-4aed-9088-daa4d7e5613f
Gill, R.
95656ecb-604f-425e-ac66-7dd36e47d94d
Thompson, D.
f02914bc-43d6-4944-91f6-6ee85e697788
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0
Wood, S.P.
430faabf-7f5c-4cf6-9bcc-5955f5e09566
Goodwin, P.M.
dec83d34-42f9-421a-91c8-4f5147c3ab97
Anthony, C.
6851d062-3caf-43dd-8e10-06dd25684146
Afolabi, P.R.
757e7f01-664c-493e-bc51-c6a2c933dc22
Mohammed, F.
e6dd5a98-bea5-484a-873a-da499c034594
Amaratunga, K.
6b6df5e8-b731-4c34-b1b8-8fc765fa1459
Majekodunmi, O.
c6df2337-0556-48e7-b390-b3d040d501d9
Dales, S.L.
a2c11795-0632-4aed-9088-daa4d7e5613f
Gill, R.
95656ecb-604f-425e-ac66-7dd36e47d94d
Thompson, D.
f02914bc-43d6-4944-91f6-6ee85e697788
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0
Wood, S.P.
430faabf-7f5c-4cf6-9bcc-5955f5e09566
Goodwin, P.M.
dec83d34-42f9-421a-91c8-4f5147c3ab97
Anthony, C.
6851d062-3caf-43dd-8e10-06dd25684146

Afolabi, P.R., Mohammed, F., Amaratunga, K., Majekodunmi, O., Dales, S.L., Gill, R., Thompson, D., Cooper, J.B., Wood, S.P., Goodwin, P.M. and Anthony, C. (2001) Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L). Biochemistry, 40 (33), 9799-9809. (doi:10.1021/bi002932l).

Record type: Article

Abstract

Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 Å resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome cL, which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.

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Published date: 1 August 2001

Identifiers

Local EPrints ID: 56195
URI: https://eprints.soton.ac.uk/id/eprint/56195
ISSN: 0006-2960
PURE UUID: 81b3c25b-892b-4f70-997f-bea0e58b0869
ORCID for P.R. Afolabi: ORCID iD orcid.org/0000-0002-0553-1578

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Date deposited: 06 Aug 2008
Last modified: 20 Jul 2019 01:17

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Contributors

Author: P.R. Afolabi ORCID iD
Author: F. Mohammed
Author: K. Amaratunga
Author: O. Majekodunmi
Author: S.L. Dales
Author: R. Gill
Author: D. Thompson
Author: J.B. Cooper
Author: S.P. Wood
Author: P.M. Goodwin
Author: C. Anthony

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