The assimilation of carbon from methane sulphonate

7.2.1 Introduction. Carbon assimilation in methylotrophs

Bacteria with the ability to grow on compounds are unified by common elements in their biochemistry. All the organisms studied assimilated carbon to cell carbon by variations on three pathways (figure 7.2.1). These pathways are named after the acceptor molecule and are the serine pathway, the ribulose monophosphate (RuMP) pathway and the ribulose bisphosphate (RuBP) pathway. A fourth pathway, found in methylotrophic yeasts, is a variant of the RuMP cycle, and is known as the XuMP pathway and will not be discussed in connection with bacterial metabolism. The diagrams illustrating the following pathways are found on page 151.

7.2.1.1 The serine pathway

The serine pathway was deduced from the short-term

incubation of 1 4C-labelled substrates, (Large at al 1961; 1962a; 1962b) with Mathylobacterium axtorquans strain AMI. The enzymes of the pathway are partly similar to those of the photoresparitory glycolate pathway (Izumi at al 1990b)

and essentially consist of a hydroxymethylation of glycine with formaldehyde to yield serine, a transfer of the amino group of serine to yield hydroxy pyruvate and the

regeneration of glycine with the production of phosphoglycerate (see figure 7.2.1.1). This C3

phosphorolated compound can then enter the tricarboxylic acid cycle, to furnish the bacterium with cellular

constituents.

The sequence and identity of enzymes in the serine cycle seems to vary little between organisms. Some differences have been shown to exist in the method by which acetyl CoA is regenerated. The involvement of isocitrate lyase and tricarboxylic acid cycle enzymes (as shown in figure 7.2.l .l ) in this process has been shown in methylotrophs such as Pseudomonas MA, Pseudomonas aminovorans, Pseudomonas MS and organism 5H2 (Anthony, 1982). However,

Methylobacterium extorquens AMI possesses no demonstrable isocitrate lyase activity, although the oxidation of acetyl CoA does result in the production of glyoxylate (Anthony, 1982). Some controversy has arisen over the validity of the only proposed route for these "icl“" variant of the serine pathway, which at present remains unresolved.

7.2.1.2 The ribulose bisphosphate (RuBP) pathway

The RuBP cycle, or Benson-Calvin cycle, of autotrophic C02 assimilation has long been recognised in autotrophic bacteria, phototrophic bacteria, cyanobacteria and green plants. More recently, some bacteria, that could be called

autotrophic methylotrophs, have also been shown to possess the RuBP pathway. The Cj growth substrate is oxidised to water and C02 , providing energy in the form of NAD(P)H. The C02 is fixed using the enzyme RUBISCO (ribulose-1,5-

bisphosphate carboxylase/oxygenase).

The Benson-Calvin cycle regenerates ribulose-1,5-

bisphosphate, synthesizing triose phosphate from 3 C02 . The phosphate is used in biosynthesis of cell material (see figure 7.2.1.2). This mode of metabolism is not absolute, varying from organism to organism, but ribulose bisphosphate is always regenerated.

The fixation of C02 can be summarised as an equation, written as follows:

6 N A D (P )H + 3C02 + 9 ATP ---> 3-phosphoglyceraldehyde

+ 6 N A D ( P ) + + 9 A D P + 8 Pj^

Organisms that gain carbon in this way include Thiobacillus versutus (Kelly and Wood, 1984) and Paracoccus denitrificans (van Verseveld and Stouthamer 1978).

7.2.1.3 The ribulose monophosphate pathway

The ribulose monophosphate cycle of HCHO fixation was also proposed by Quayle (1965). Since the first version of the pathway, variants have been identified, mostly in obligate methanotrophs and roethanolotrophs (Figures 7.2.1.3 and 7.2.1.4.). Common features in all the variants are the synthesis of D-erythro-L-glycero-3-hexulose-6-phosphate

(known as hexulose-6-phosphate for the sake of brevity) from

ribulose-5-phosphate and formaldehyde, and the isomerisation of hexulose-6-phosphate to form fructose-6-phosphate. The variation between organisms lies in the regeneration of ribulose-5-phosphate.

7.2.1.4 The concept of key enzymes

It was at first suggested that only one pathway of the three functioned in any given species of bacterium (Anthony 1975; Quayle, 1972), but work on Pseudomonas oxalaticus (Quayle, 1961) and work mentioned by Colby et a l . (1979) on

Pseudomonas gazotropha showed that one Cj^ assimilation pathway may be used by an organism to enable growth on one compound, and another pathway may be used for a second compound. No convincing evidence has been presented to show the simultaneous operation of two or more pathways.

The presence of a particular assimilation pathway in a bacterium has been classically determined by the presence of one or two key enzymes, taken as being essential to the functioning of the pathways. Hydroxypyruvate reductase (HPR, see figure 7.2.1.1) has been taken as an indicator of the presence of the serine pathway, hexulose phosphate synthase (HPS, see figure 7.2.1.3 and 7.2.1.4) an indicator of the RuMP pathway and both RUBISCO and phosphoribulokinase an indicator of a RuBP pathway. However, the presence of an enzyme is no absolute indicator of the method of carbon assimilation.

The methanotroph Methylococcus capsulatus (Bath) has RUBISCO and phosphoribulokinase, despite having all the enzymes of

C 1 amines

Figure 7.2.1 Assinilatory and dissiiilatory pathways in Mthylotrophs (After latnan, 1980)

(D

Figure 7.2.1.1 The serine pathway for the assimilation of carbon during netbylotrophic growth.

Figure 7.2.1.2 Tbe assimilation of carbon by aethylotrophic autotrophs. Encircled nuabers indicate the stoichioaetry of tbe transformations. PQQ • pyrrologuinoline quinone. PQQB2 * reduced pyrroloquinoline quinone

3^