MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 5. MICROBIAL METABOLISM

MECHANISMS OF CARBON ASSIMILATION IN MICROORGANISMS

Carbon sources for microorganisms include both organic and inorganic substances (carbon dioxide, carbonates).

Assimilation of inorganic carbon. Carbon dioxide is the most abundant form of carbon on Earth. Many microorganisms, known as autotrophs, have The ability to incorporate CO₂ into cellular carbon. This process is referred to as CO₂ fixation.

Carbon dioxide fixation. Microorganisms utilize four distinct pathways for carbon dioxide fixation:

1. The Calvin Cycle (reductive Pentose Phosphate Pathway or ribulose biphosphate pathway, RuBP);

2. The reductive Tricarboxylic Acid Cycle (Evans-Buchanan-Arnon cycle, rTCA);

3. The reductive acetyl-CoA pathway (Wood-Ljungdahl pathway, rACA);

4. The 3-hydroxypropionate cycle.

The Calvin cycle (Fig. 5.30) is The most significant biosynthetic pathway on Earth. It is utilized by the majority of photosynthetic organisms, including plants, cyanobacteria, purple Bacteria, and green bacteria. The Calvin cycle consists of three phases:

1. Carbon dioxide fixation. CO₂ is coupled with ribulose-1,5-bisphosphate, a five-carbon compound, yielding two molecules of 3-phosphoglyceric acid. One of the two carboxyl groups in this compound originates from CO₂. The reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo), the signature key enzyme of this cycle.

Class="center">Ribulose-1,5-bisphosphate + CO2 + H2O → 3-Phosphoglyceric acid (2 molecules)

2. Reduction of phosphoglyceric acid carboxyl groups to aldehydes. The carboxyl group of 3-phosphoglyceric acid is first activated via phosphorylation to form 1,3-diphosphoglyceric acid, which is subsequently reduced to 3-phosphoglyceraldehyde. This stage consumes virtually all the energy and reducing equivalents required for CO₂ fixation. A portion of the 3-phosphoglyceraldehyde is converted into fructose-6-phosphate to fuel biosynthetic processes.

3-Phosphoglyceric acid + ATP → 1,3-Diphosphoglyceric acid + ADP

1,3-Diphosphoglyceric acid + NADPH₂ → 3-Phosphoglyceraldehyde + NADP⁺ + P_i

3. Regeneration of the CO₂ acceptor. The remaining Reactions of the cycle regenerate ribulose-1,5-bisphosphate. These begin with the synthesis of fructose-6-phosphate from two triose phosphate molecules. This is followed by a rearrangement of the carbon Skeleton to generate pentose-5-phosphates, mirroring the reverse (reductive) reactions of The pentose phosphate pathway. The regeneration of the CO₂ acceptor is completed by the ATP-dependent phosphorylation of ribose-5-phosphate to ribulose-1,5-bisphosphate. This process is catalyzed by the second specific key enzyme of the Calvin cycle, phosphoribulokinase.

Energy balance of the Calvin cycle. To generate a single net molecule of fructose-6-phosphate—which can then be withdrawn from the cycle for Biosynthesis—six molecules of ribulose-1,5-bisphosphate must be carboxylated, meaning that 6CO₂ are fixed. Following carboxylation, these six molecules of ribulose-1,5-bisphosphate are converted into 12 molecules of 3-phosphoglyceric acid, then into 12 molecules of 1,3-diphosphoglyceric acid, and finally reduced to 12 molecules of 3-phosphoglyceraldehyde. The Cell expends 12 ATP and 12 NADPH₂ on these transformations. An additional 6 ATP are consumed during the regeneration of the CO₂ acceptor to phosphorylate ribose-5-phosphate into ribulose-1,5-bisphosphate. Thus, the synthesis of a single glucose molecule "costs" the cell 18 ATP and 12 NADPH₂. The overall stoichiometric equation for CO₂ fixation via the Calvin cycle is as follows:

6CO₂ + 12 NADPH₂ + 18 ATP → C₆H₁₂O₆ + 12 NADP+ + 18 ADP + 18 P_i

Fig. 5.30. The Calvin cycle

(reductive pentose phosphate pathway or ribulose bisphosphate pathway)

The reductive tricarboxylic acid cycle was described by Evans, Buchanan, and Arnon in 1966 in the green sulfur bacterium Chlorobium thiosulfatophilum. It was later discovered in certain sulfate-reducing bacteria and thermophilic hydrogen-oxidizing bacteria of the genus Hydrogenobacter. The reductive TCA cycle is essentially a modified Krebs cycle operating in reverse, in a reductive direction (Fig. 5.31).

Fig. 5.31. Reductive tricarboxylic acid cycle (synonyms: Evans-Buchanan-Arnon cycle, reductive TCA cycle)

There are full and short reductive TCA cycles. As a result of one full turn of the cycle, four molecules of СО₂ are fixed and OAA is synthesized. Since OAA itself is an intermediate product of the cycle, after one rotation, one molecule of OAA is regenerated, while a second one is synthesized from four fixed and reduced СО₂ molecules. During a short cycle, two fixed СО₂ molecules yield one molecule of acetate.

As can be seen from the figure, the full and short cycles share the same sequence of reactions from OAA to citrate. That is, starting from OAA, a complete Rotation of the short reductive TCA cycle leads to OAA regeneration and the synthesis of acetate from СО₂. Ultimately, it is the short reductive TCA cycle—which produces acetyl-CoA from 2СО₂—that is the reverse of the Krebs cycle. The full reductive TCA cycle involves four СО₂ fixation and reduction reactions:

1. Acetyl-CoA + СО₂ + Ferredoxin_red → Pyruvate + CoA + Ferredoxin_ox

2. Succinyl-CoA + СО₂ + Ferredoxin_red → α-Ketoglutarate + CoA + Ferredoxin_ox

3. α-Ketoglutarate + СО₂ + NADPH₂ → Isocitrate + NADP⁺

4. Phosphoenolpyruvate + СО₂ + → OAA + P_i

Reactions (2) and (3) occur in both the standard Krebs cycle and the reductive TCA cycle, whereas reactions (1) and (4) are unique to the reductive TCA cycle. Reactions (1) and (2) are driven by ferredoxins rather than NADPH₂. СО₂ assimilation requires Energy Expenditure. The Discovery of the Evans–Buchanan–Arnon cycle confirmed the evolutionary link between heterotrophic and photoautotrophic microorganisms.

The reductive acetyl-CoA pathway is found in the METABOLISM of autotrophic sulfate reducers, homoacetogens, and methanogens. Unlike other СО₂ assimilation pathways, it is non-cyclic (Fig. 5.32). One molecule of СО₂ is bound by a special cofactor, tetrahydrofolic acid, and reduced to a methyl group. A second СО₂ molecule is reduced to a carbonyl (C=O) group by the enzyme carbon monoxide dehydrogenase. The methyl and carbonyl groups are then combined into acetyl-CoA, which is utilized in further biosynthetic processes. Two molecules of СО₂ are reduced at the expense of 4Н₂.

Overall equation of the process: 4Н₂ + 2СО₂ = Acetate + 2Н₂О

The acetyl-CoA pathway can also operate in the reverse, oxidative direction. Heterotrophic sulfate reducers employ the oxidative acetyl-CoA pathway for acetate Cleavage (Fig. 6.26).

The 3-hydroxypropionate СО₂ fixation cycle (Fig. 5.33) was discovered in green non-sulfur photosynthetic bacteria of the genus Chloroflexus and certain archaea.

A full turn of this cycle fixes 2СО₂ and synthesizes glyoxylate (CHOCOOH). The first СО₂ molecule is accepted by acetyl-CoA to form malonyl-CoA, while the second СО₂ molecule combines with propionyl-CoA to yield methylmalonyl-CoA. The terminal intermediate of the cycle, malyl-CoA, is cleaved into acetyl-CoA and glyoxylate. Acetyl-CoA fixes СО₂ anew in the next turn, whereas glyoxylate is channeled into biosynthesis. The process can be described by the following equation:

2НСО₃^- + 2 NADPH₂ + 3Н⁺ + 3 ATP → CHO-COO^- + 2 NADP⁺ + 3 ADP + 3 P_i

Fig. 5.32. Reductive acetyl-CoA pathway (synonym: Wood–Ljungdahl pathway): THF, tetrahydrofolic acid

Fig. 5.33. 3-Hydroxypropionate cycle

The electron Donors and The Nature of many Enzymes involved in this cycle remain unstudied.

Formaldehyde assimilation. Methylotrophic microorganisms utilize three pathways for formaldehyde (_СН2О) assimilation:

1. Serine pathway.

2. Ribulose monophosphate cycle (hexulose phosphate pathway, RuMP).

3. Dihydroxyacetone pathway.

The first two pathways are typical of methylotrophic bacteria, whereas the last one is restricted to methylotrophic Yeasts.

The serine pathway is a cyclic process whose end product is acetyl-CoA, synthesized from one molecule of formaldehyde and one molecule of СО₂ (Fig. 5.34).

Fig. 5.34. The serine pathway of formaldehyde assimilation

The first reaction of this cycle involves the interaction of formaldehyde (C₁) with Glycine (C₂) to form serine (C₃). This reaction is catalyzed by hydroxymethyltransferase, which requires tetrahydrofolic acid (THF) as a cofactor. The process proceeds via THF-bound intermediates. When formaldehyde attaches to the cofactor of hydroxymethyltransferase, 5,10-methylenetetrahydrofolic acid (methylene-

THF) is formed. Through reaction (1) (Fig. 5.34, 1), formaldehyde is transferred from methylene-THF to glycine, yielding serine.

In the subsequent reactions (Fig. 5.34, 2), an amino group is transferred from serine to glyoxylate. As a result, serine is converted into hydroxypyruvate, while glyoxylate is converted into glycine. Glycine then reacts with another formaldehyde molecule, and hydroxypyruvate undergoes further transformation (Fig. 5.34, 3).

Hydroxypyruvate is reduced to glyceric acid, which is subsequently phosphorylated to 2-phosphoglycerate with the consumption of 1 ATP. At this point, the serine pathway branches: a portion of the 2-phosphoglyceric acid is converted into 3-phosphoglyceric acid for biosynthesis, while the remainder is transformed into phosphoenolpyruvate (PEP). PEP (C₃) is carboxylated with CO₂ to form oxaloacetate (C₄), which is reduced to malate and converted into malyl-CoA using ATP. Catalyzed by malyl-CoA lyase, malyl-CoA is cleaved into glyoxylate (a C₂ compound), which re-enters the cycle, and acetyl-CoA (a C₂ compound), which is channeled into biosynthesis.

The ribulose monophosphate cycle (Fig. 5.35) is a cyclic process of formaldehyde assimilation that consists of three stages:

1. Fixation. Ribulose-5-phosphate serves as the formaldehyde acceptor. Formaldehyde and ribulose-5-phosphate condense in a reaction catalyzed by hexulose phosphate synthase to yield hexulose-6-phosphate, which is then converted into fructose-6-phosphate by the action of hexulose phosphate isomerase. Hexulose phosphate synthase and hexulose phosphate isomerase are the only unique enzymes specific to this pathway. The remaining steps are catalyzed by Enzymes of the Entner-Doudoroff Pathway.

2. Cleavage. Fructose-6-phosphate is cleaved into C₃ compounds via one of two routes:

a) fructose-6-phosphate is phosphorylated to fructose-1,6-diphosphate and subsequently cleaved into dihydroxyacetone phosphate and phosphoglyceraldehyde;

b) fructose-6-phosphate is first isomerized to glucose-6-phosphate, which is converted into D-glucono-1,5-lactone-6-phosphate, then into 6-phosphogluconate, and finally into 2-keto-3-deoxy-6-phosphogluconate (KDPG). KDPG is cleaved into 3-phosphoglyceraldehyde and pyruvate by KDPG aldolase.

One of the phosphotrioses (dihydroxyacetone phosphate or pyruvate) is directed toward biosynthesis.

3. Rearrangement. The final stage of the process is the rearrangement of phosphotrioses, which regenerates the formaldehyde acceptor, ribulose-5-phosphate. This can occur via several pathways, but overall, two molecules of xylulose-5-phosphate and one molecule of ribose-5-phosphate are converted into three molecules of ribulose-5-phosphate, completing the cycle.

Thus, as a result of the ribulose monophosphate cycle, three molecules of formaldehyde yield a triose (dihydroxyacetone phosphate or pyruvate), which is channeled into biosynthesis.

The overall equation of the process:

3CH₂O + 2 ATP → Phosphotriose + 2 ADP + P_i

Fig. 5.35. Scheme of the ribulose monophosphate cycle for formaldehyde assimilation (synonym: hexulose phosphate pathway, RuMP)

* - reactions that can occur only when at least three molecules of formaldehyde are incorporated into the process

Assimilation of C₂ substrates by microorganisms. When growing on media containing acetate as the sole carbon source, aerobic microorganisms assimilate acetyl-CoA via The Glyoxylate cycle.

The glyoxylate cycle is a sequence of reactions that converts two acetyl units into one C₃ compound with the decarboxylation of the fourth carbon atom: 2C₂ → 1C₃ + 1C₁ (Fig. 5.36).

Fig. 5.36. The glyoxylate cycle of acetyl-CoA assimilation in microorganisms

In the first step of this cycle, acetyl-CoA condenses with oxaloacetate to form citrate, which is subsequently converted into isocitrate. Isocitrate is then cleaved by isocitrate lyase into two intermediates: glyoxylate and succinate.

Isocitrate → Glyoxylate + Succinate

In the second stage, two molecules of malate are synthesized. One molecule is formed directly from succinate via fumarate, while the second is produced through the Condensation of the glyoxylate generated in the first step with a second molecule of acetyl-CoA. This reaction is catalyzed by malate synthase:

Glyoxylate + Acetyl-CoA + H₂O → Malate + CoA-SH

In the Third Stage, two molecules of malate yield two molecules of OAA, one of which is channeled into biosynthesis while the other Functions within the glyoxylate cycle. To enter biosynthetic pathways, OAA is decarboxylated to pyruvate or phosphoenolpyruvate.

Gluconeogenesis is the metabolic pathway through which glucose is synthesized from pyruvate (Fig. 5.37). While it shares many steps with Glycolysis—the process by which glucose is oxidized to pyruvate—these reactions proceed in the reverse (reductive) direction.

However, gluconeogenesis and glycolysis also exhibit several key differences. Unlike glycolysis, which yields 2 ATP during The oxidation of glucose, the Synthesis of glucose via gluconeogenesis consumes 6 ATP.

Fig. 5.37. Pathway of gluconeogenesis

Glycolysis:

Glucose + 2 NAD⁺ + 2 ADP + 2 P_i → 2 Pyruvate + 2 NADH₂ + 2 ATP + 2H₂O

Gluconeogenesis:

2 Pyruvate + 2 NADH₂ + 4 ATP + 2 GTP + 6H₂O → Glucose + 4 ADP + 2 GDP + 6 P_i + 2 NAD⁺ + 2H⁺ (GTP and ATP are mutually interconvertible: GDP + ATP → GTP + ADP). While glycolysis converts phosphoenolpyruvate into pyruvate in a single step catalyzed by pyruvate kinase with the net production of 1 ATP, gluconeogenesis converts pyruvate back into phosphoenolpyruvate via a two-step process involving an OAA intermediate, consuming 1 ATP and 1 GTP and requiring two separate enzymes: pyruvate carboxylase and phosphoenolpyruvate carboxykinase.