Metabolism of Cad16 T in absence of light

One of the aims of the proteomic analysis was to elucidate the metabolic process that allowed strain Cad16^T to fix CO2 in the dark. Among the 17 protein spots that were significantly up-regulated in the dark, three in particular are of interest due to their potential role in the autotrophic dicarboxylate-hydroxybutyrate (DC/HB) cycle (e.g., enzymes 4, 21 and 22 in Figure 7). This cycle was recently discovered in the hyperthermophilic Archaeum Ignicoccus hospitalis by Huber et al. (Huber et al. 2008), and is presumed to be present only in Archaea. The cycle converts acetyl-CoA and two inorganic carbons (CO2) to succinyl-CoA, using essentially the same enzymes as the rTCA cycle (see Figure 7 B). The carboxylases involved in this cycle, which are responsible for inorganic carbon fixation, are pyruvate synthase and phosphoenolpyruvate (PEP) carboxylase. After the inorganic carbon fixation is complete, the acetyl-CoA must be regenerated. First, the CO₂ fixation product succinyl-CoA is reduced to 4-hydroxybutyrate, which is activated to 4-hydroxybutyryl-CoA and then dehydrated to crotonyl-CoA by 4-hydroxybutyryl-crotonyl-CoA dehydratase. 4-hydroxybutyryl-crotonyl-CoA dehydratase is considered a key enzyme in this DC/HB cycle. Crotonyl-CoA is further modified to (S)-3-hydroxybutyryl-CoA, then acetoacetyl-CoA and finally to two acetyl-CoA molecules. The DC/HB cycle generates an additional molecule of acetyl-CoA (see Figure 7 A) and overall uses eight ATP molecules and 10 reducing factors to fix H2CO3 and CO2 into a triose phosphate (glyceraldehyde-6-phosphate). A similar cycle, the hydroxypropionate/hydroxybutyrate (HP/HB) cycle (see Figure 7 B) that was described in the aerobic autotrophic Sulfolobales (Archaea), involves another set of carboxylases for fixing CO2 (the acetyl-CoA/propionyl-CoA carboxylase).

Figure 7. (A) The dicarboxylate/4-hydroxybutyrate cycle described in Desulfurococcales and Thermoproteales;

(B) the 3-hydroxypropionate/4-hydroxybutyrate cycle described in Sulfolobales.

Enzymes: 1, pyruvate synthase; 2, pyruvate:water dikinase; 3, PEP carboxylase; 4, malate dehydrogenase; 5, fumarate hydratase; 6, fumarate reductase (natural electron acceptor is not known); 7, succinyl-CoA synthetase; 8, acetyl-CoA/propionyl-CoA carboxylase; 9, malonyl-CoA reductase; 10, malonic semialdehyde reductase; 11, 3-hydroxypropionate-CoA ligase; 12, 3-hydroxypropionyl-CoA dehydratase; 13, acryloyl-CoA reductase; 14, methylmalonyl-CoA epimerase; 15, methylmalonyl-CoA mutase; 16, succinyl-CoA reductase; 17, succinic semialdehyde reductase; 18, 4-hydroxybutyrate-CoA ligase; 19, 4-hydroxybutyryl-CoA dehydratase; 20, crotonyl-CoA hydratase; 21, (S)-3-hydroxybutyryl-crotonyl-CoA dehydrogenase (NAD⁺); 22, acetoacetyl-CoA-ketothiolase. (Berg 2011).

One of the key enzymes in the DC/HB cycle is the 4-hydroxybutyryl-CoA dehydratase. This enzyme is considered a “radical enzyme”, as it uses radicals as intermediates during the metabolic reactions. Because they are highly reactive towards dioxygen, radicals are often used in catabolic reactions in anaerobic micro-organisms (Buckel and Golding 1998; Matias et al.

2005; Buckel and Golding 2006). Among the 17 proteins that were up-regulated in the dark,

three enzymes involved in anti-oxidant stress responses such as a superoxide dismutase and two peroxiredoxin were identified (see Table 2 in the research paper 3). Thus, the enhanced activity of the 4-hydroxybutyryl-CoA dehydratase in the dark could result in the production of harmful free radicals that could be inactivated by an increased expression of antioxidant enzymes.

The HB/DC cycle could be hypothetically separated in two independent part, the dicarboxilate and the hydroxybutyrate part. The first is composed of the same enzymes of the initially part of the rTCA cycle (see Figure 8 A), where our up-regulated enzyme (number 2, malate deshydrogenase) reversely transform the oxaloacetate in malate. In others words, this enzyme could be involved in the hypothetical DC/HB cycle but also in the TCA or rTCA cycle, producing ATP and reducing power or fixing CO2 respectively. In the other hand, two others proteins up-regulated in the dark corresponding to the last steps of the DC/HB cycle, which participate also in the PHB degradation pathway (see Figure 8 B). This enzymes reversely catalyzed the final steps of the PHB granules degradation (see Figure 8 B, enzymes PhaB: 3-ketoacyl-ACP reductase and PhaA: acetoacetyl-CoA-ketothiolase). Interestingly, in presence of light the up-regulation of three enzymes involved in the synthesis of PHB globules was shown, which presumes a high concentration of reserve globules in the bacterial cell. How said before, the degradation of the PHB granules appears to produce acetyl-CoA and increase the overall reducing power, such as concentration of available NADPH. In purple non-sulfur bacteria was shown that the form II of the RuBisCO enzyme is commonly involved in the process of CO₂ fixation as an electron sink for reducing equivalents derived from the oxidation of reserve compounds (Dubbs and Robert Tabita 2004; Joshi et al. 2009; Laguna et al. 2010). A similar operon structure as that found in purple non-sulfur bacteria is also present in the draft genome of strain Cad16^T, with two distinct regions harboring genes coding for RuBisCO form I and form II.

So we could suppose that the reducing power resulting from the PHB degradation can be utilized in the process of dark CO2 fixation. Moreover, the excess in acetyl-CoA is most probably used to generate additional reducing power via the TCA or glyoxylate cycle. In both of these cycles, one of our up-regulated enzymes (malate dehydrogenase) reversibly catalyzes the oxidation of malate to oxaloacetate by reducing NAD⁺ to NADH.

Figure 8. (A) The reductive citric acid (rTCA) cycle. The pathway of acetyl-CoA assimilation to pyruvate, phosphoenolpyruvate (PEP), and oxaloacetate is also shown (Berg 2011). (B) For deviations from this variant of the cycle, see the text. (B) Cyclic metabolism of PHB biosynthesis and degradation in bacteria (Sudesh et al. 2000).

Enzymes rTCA: 1, ATP-citrate lyase; 2, malate dehydrogenase; 3, fumarate hydratase; 4, fumarate reductase (natural electron donor is not known); 5, succinyl-CoA synthetase; 6, ferredoxin (Fd)-dependent b2-oxoglutarate synthase; 7, isocitrate dehydrogenase; 8, aconitate hydratase; 9, Fd-dependent pyruvate synthase; 10, PEP synthase;

11, PEP carboxylase.

Enzymes PHB synthesis/degradation: PhaA, acetoacetyl-CoA-ketothiolase; PhaB, 3-ketoacyl-ACP reductase;

PhaC, PHA synthase; PhaZ, PHA depolymerase; 1, dimer hydrolase; 2, (R)-3-hydroxybutyrate dehydrogenase; 3, acetoacetyl-CoA synthetase; 4, NADH-dependent acetoacetyl-CoA reductase.