The oxidation of methanol in methylotropic bacteria
The oxidation of methanol in methylotropic bacteria
Methylobacterium AM1 and Methylophilus methylotrophus are known to contain modifier proteins with the abiilty to alter the substrate specificity of methanol dehydrogenase. Both M-proteins have been reported to be soluble colourless proteins of native molecular weight 130kD, containing two identical subunits and constituting up to 6% of the soluble cell protein (Page and Anthony, 1986). This thesis reports the purification of the M-protein of M.methylotrophus by the method used previously with an additional final step which showed that the active component of the M-protein preparation was not the major protein, containing two 67kD subunits (previously designated as the M-protein band) but a minor contaminant of this protein. The active components could not be purified to homogeneity. Paracoccus denitrificans was shown also to contain a modifier protein similar to those characterised previously in terms of native molecular weight and ability to inhibit formaldehyde oxidation by MDH in a dye-linked assay at pH 9. However, its subunit molecular weight was 45kD and the quantity of this protein in the cell was much less than that of MDH. It was demonstrated that in P.denitrificans M-protein was localised exclusively within the periplasmic space of the bacterium using a lysozyme/EDTA treatment to produce spheroplasts. In order to study the effect of the M-protein from P.denitrificans upon MDH under physiological conditions it was necessary to set up a cytochrome c-linked assay, such as that described by Beardmore-Gray and Anthony (1983). Since the cytochrome c acting as electron acceptor from MDH was unknown in this organism, all cytochromes c present during methylotrophic growth were purified and tested for their ability to accept electrons from MDH. The soluble 22kD cytochrome c552 was found to be the physiological electron acceptor for MDH in P.denitrificans, contrary to previous speculation that the 30kD c553 performed this role. Cytochrome c552 was used to mediate electron flow between MDH and horse-heart cytochrome c in a physiological assay at pH 7.5. In this assay M-protein inhibited the oxidation of formaldehyde. It also diminished rates of methanol oxidation while increasing the affinity of MDH for methanol. Thus it is proposed that M-protein may have a role both in the regulation of formaldehyde and methanol oxidation. In both cases the greatest effect is observed when the molar ration of MDH to M-protein in the assay was 5:1; however, this molar ratio was much greater in periplasmic protein extracts, at least under the single set of conditions used for the growth of the bacteria, suggesting that there may be too little M-protein expressed to perform the role(s) proposed. During these studies two strains of P.denitrificans 8944 were used: one obtained from Oxford University, the other form the N.C.I.B. Although both were identical with respect to their expression of M-protein, the Oxford strain expressed two novel cytochrome c complexes during methylotrophic and heterotrophic growth, both of which were purified and characterised. The major complex (up to 25% of total cytochrome c) was shown to be a heterodymeric cytochrome c containing subunits of 30kD and 15kD. The large subunit contained three high spin haems and the small subunit two low spin haems. The functions of either complex are unknown.
University of Southampton
1990
Long, Anthony Richard
(1990)
The oxidation of methanol in methylotropic bacteria.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Methylobacterium AM1 and Methylophilus methylotrophus are known to contain modifier proteins with the abiilty to alter the substrate specificity of methanol dehydrogenase. Both M-proteins have been reported to be soluble colourless proteins of native molecular weight 130kD, containing two identical subunits and constituting up to 6% of the soluble cell protein (Page and Anthony, 1986). This thesis reports the purification of the M-protein of M.methylotrophus by the method used previously with an additional final step which showed that the active component of the M-protein preparation was not the major protein, containing two 67kD subunits (previously designated as the M-protein band) but a minor contaminant of this protein. The active components could not be purified to homogeneity. Paracoccus denitrificans was shown also to contain a modifier protein similar to those characterised previously in terms of native molecular weight and ability to inhibit formaldehyde oxidation by MDH in a dye-linked assay at pH 9. However, its subunit molecular weight was 45kD and the quantity of this protein in the cell was much less than that of MDH. It was demonstrated that in P.denitrificans M-protein was localised exclusively within the periplasmic space of the bacterium using a lysozyme/EDTA treatment to produce spheroplasts. In order to study the effect of the M-protein from P.denitrificans upon MDH under physiological conditions it was necessary to set up a cytochrome c-linked assay, such as that described by Beardmore-Gray and Anthony (1983). Since the cytochrome c acting as electron acceptor from MDH was unknown in this organism, all cytochromes c present during methylotrophic growth were purified and tested for their ability to accept electrons from MDH. The soluble 22kD cytochrome c552 was found to be the physiological electron acceptor for MDH in P.denitrificans, contrary to previous speculation that the 30kD c553 performed this role. Cytochrome c552 was used to mediate electron flow between MDH and horse-heart cytochrome c in a physiological assay at pH 7.5. In this assay M-protein inhibited the oxidation of formaldehyde. It also diminished rates of methanol oxidation while increasing the affinity of MDH for methanol. Thus it is proposed that M-protein may have a role both in the regulation of formaldehyde and methanol oxidation. In both cases the greatest effect is observed when the molar ration of MDH to M-protein in the assay was 5:1; however, this molar ratio was much greater in periplasmic protein extracts, at least under the single set of conditions used for the growth of the bacteria, suggesting that there may be too little M-protein expressed to perform the role(s) proposed. During these studies two strains of P.denitrificans 8944 were used: one obtained from Oxford University, the other form the N.C.I.B. Although both were identical with respect to their expression of M-protein, the Oxford strain expressed two novel cytochrome c complexes during methylotrophic and heterotrophic growth, both of which were purified and characterised. The major complex (up to 25% of total cytochrome c) was shown to be a heterodymeric cytochrome c containing subunits of 30kD and 15kD. The large subunit contained three high spin haems and the small subunit two low spin haems. The functions of either complex are unknown.
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Published date: 1990
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Local EPrints ID: 460518
URI: http://eprints.soton.ac.uk/id/eprint/460518
PURE UUID: 0c8fce1c-fc27-402f-a3ef-f3707a0ba480
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Date deposited: 04 Jul 2022 18:23
Last modified: 04 Jul 2022 18:23
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Author:
Anthony Richard Long
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