7.3.2 Methane as a primary product in the degradation of
MSA (Scheme 1)
A close structural analogue of MSA, methane phosphonate (MPA), is used as a phosphorus source by several
organisms including Escherechia coli and Pseudomonas testosteroni (See section 6.1). The compound is not assimilated in this heterotroph, as methane is released but the organisms can derive phosphate from the process. A similar process could be suggested for M2 growing on MSA. Methane, rather than being released could act as a source of cell carbon, its oxidation providing energy. Sulphate would be the other product of the initial cleavage.
The main evidence against the cleavage of methane from MSA playing any part in the oxidation of MSA in M2 was three-fold. Methane was not a growth substrate for M2
(see table 6.3.3.1), nor an oxidation substrate of MSA- grown cells and it could not be detected at any time during the organism's growth cycle by GC. The generation of methane as a degradative route for MSA will not be discussed further in this thesis.
7.3.3 Methanol as a primary product in the degradation of
MSA (Scheme 2)
The methane sulphonate molecule has its weakest bond connecting the carbon and sulphur atoms. If this bond is the site of the first enzymatic cleavage and the products would be a methyl group and a sulphonate group, attached
to some other molecule or atom. The subsequent steps in the oxidation of these groups would lead to methanol and sulphite.
7.3.4 Formaldehyde as a primary product in the
degradation of HSA (Scheme 3)
A monooxygenic enzyme catalysed attack on the methane sulphonate molecule could lead to the direct formation of formaldehyde (figure 7.3.4).
Figure 7.3.4 The aetabolisa of HSA to yield fonaldehyde as a priiary product.
Formaldehyde might then be subjected to further oxidation to formate, then C02 and water, or be used for
assimilation into cell carbon. This pathway uses the same amount of oxygen to effect complete mineralisation of MSA as scheme 2, but may conceivably not result in the
production or use of the same cofactors. The model that results in the production of methanol is conceptually
H 0 Mathanaaulphonlcadd H O II II O H— C 0 H H O S — OH II
o
similar to the methanol and methane oxidation pathways found in many methylotrophs and methanotrophs
respectively, the oxidation from the level of methanol producing resulting in the formation of reducing power at each stage of the mineralisation. The amount of reducing power generated from the direct oxidation of MSA to formaldehyde may differ from the conventional pathway, but that from the mineralisation of formaldehyde would be the same.
7.3.5 Oxidation of a carrier molecule side-chain (Scheme 4)
Methylotrophs such as Pseudomonas MS (Kung and Wagner, 1970), accomplish the oxidation of compounds containing methyl groups, such as trimethyl sulphonium chloride, by transferring the group to a carrier molecule. In the case of Pseudomonas MS this molecule is tetrahydrofolate. The methyl group is then oxidised as a side chain of the larger carrier molecule, and released from it as formate, which can then be oxidised to C02 and water. The carrier molecule with an aldehyde side chain can also be fed directly into, for example, the serine pathway of carbon assimilation as N 5 •1 0methlyenetetrahydrofolate. Such a system could allow the oxidation of the methyl group of MSA, after it had been split from the sulphonate moiety. Equally, a carrier molecule could be used to accept the whole molecule:
Carrier + C H3so3Na --- > Carrier-CH2S03Na
This would allow the oxidation of the side chain to proceed via either of the pathways outlined in sections 7.3.3 and 7.3.4. Thus the questions surrounding the route of oxidation of MSA can be applied to hypothetical
pathways involving free or bound methyl and aldehyde groups, without elementary identification of the identity of the putative carrier molecule.
7.3.6 Comments
The work on M2 has, for the major part, been directed towards elucidating the pathway of MSA oxidation, whether by scheme 2 or scheme 3. Since neither compound could be detected in media during growth of M2 on MSA, two
approaches have been taken to induce the accumulation of an identifiable intermediate. These have been to inhibit the methanol-oxidising activity of MSA-grown cells and to observe the oxidation of MSA analogues by MSA-grown
cells.
7.4 The oxidation of methane sulphonate
7.4.1 Introduction
The complete oxidation of MSA requires the involvement of two molecules of oxygen:
CH3S03Na + 202 --- > C02 + H 20 + NaHS04
If an oxygenase is involved in the initial step of the pathway, then an additional 0.5 mol oxygen is required:
CH3S03Na + 2*503 + 2 [H] --- > C02 + 2H20 + NaHS04
Stoichiometry is the amount of one substrate used compared to the amount of another substrate, in a given reaction. This parameter can be applied to the metabolism of MSA: the stoichiometry of the first reaction with regard to oxygen is 2, and in the second it is 2*5. The consumption of oxygen in the presence of a substrate can be measured using the oxygen electrode, and is calibrated by a method based on a
supposition that aerated distilled water contains 245 nmole of oxygen ml-1. The calibrated electrode can yield the amounts and rates of use of oxygen in the presence of substrates. Modes of oxidation can be deduced from
stoichiometries, based on a knowledge of the chemistry of the substrate.
7.4.2 Substrate specificity of MSA-grown cells
The methylotrophic strain M2, when grown on MSA, oxidised few compounds (see table 7.4.1).
Potential substrate analogues (ethane sulphonate, methane phosphonate, monomethyl sulphate) were not oxidised, which could lead to the hypothesis that the enzyme(s) involved in the first stages of MSA metabolism (including any involved with transport across the membrane of the cell), are
specific.
The absence of any oxygen consumption in the presence of ethylene suggests that the organism does not have the capacity for
epoxidation. This result was repeated using cell-free extracts,