BIOSYNTHETIC PATHS lead from 11 PRECURSOR METABOLITES to form ~75 "BUILDING BLOCKS." (Nitrogen and Sulfur incorporation included here.)

FUELING PATHS lead from nutrients found in medium to produce energy, reducing power, C-1 units, and precursor metabolites.

(Phosphate acquisition is included here.)

In more traditional terms: Biosynthetic pathways are referred to as ANABOLIC PATHWAYS, while fueling paths are referred to as CATABOLIC PATHWAYS. Pathways that produce both energy and precurors for biosynthesis are AMPHIBOLIC (glycolysis, TCA cycle, and pentose-phosphate pathways).

1. Glucose 6-phosphate (6-carbons)

2. Fructose 6-phosphate (6-carbons)

3. Triose phosphate (3-carbons)

4. 3-Phosphoglycerate (3-carbons)

5. Phosphoenolpyruvate (3-carbons)

6. Pyruvate (3-carbons)

7. Ribose 5-phosphate (5-carbons)

8. Erythrose 4-phosphate (4-carbons)

9. Acetyl-CoA (2-carbons)

10. a-Ketoglutarate (5-carbons)

11. Oxaloacetate (4-carbons)

Error in Book: Textbook includes Succinyl-CoA because the authors believed it to be a precursor of HEME biosynthesis for cytochromes. Actually precursor is a-ketoglutarate. Hence, not 12 precursors but only 11.

Table 1

Basic building blocks are pretty straight-forward: 20 amino acids; 8 nucleotides; a few fatty acids (typically 3-6); glycerol phosphate; LPS components; peptidoglycan components; coenzymes and prosthetic groups (~25 molecules). Total: ~75 molecules.

Biosynthetic pathways are summarized in text on pages 137-141. Complexity of pathways differs considerably. Simplest cases have a single enzyme to convert precursor into building block (e.g., pyruvate to alanine). Other pathways require 10-15 steps. Some paths branch, produce multiple products ("product families")

Survey of Fig. 3 (p. 139-141)demonstrates the primary requirements: precursors, NADH / NADPH, ammonia (NH₄⁺), sulfate (SO₄^-2), C-1 carbon units, and high-energy phosphate bonds (~P = ATP/GTP/UTP/CTP etc.).

Plants, algae, fungi and most procaryotes use sulfate as a sulfur source. Sulfate is abundant in many environments, esp. sea water. Can also be used as an electron acceptor under anaerobic conditions.

Conversion of sulfate to H₂S is an 8-electron reduction reaction. Sulfate is quite stable and cannot be acted upon until activated.

ATP + sulfate is converted to Adenosine-5'-PhosphoSulfate (APS) by ATP sulfurylase with loss of pyrophosphate (cost = 2 ATP equivalents). APS is phosphorylated again by APS kinase to form 3'-PhosphoAdenosine-5'-PhosphoSulfate (PAPS). A 2-electron reduction follows forming SO₃^-2 (sulfite) and releasing PAP. The electrons for this reduction usually come from reduced thioredoxin, which contains two Cysteine residues; the oxidized thioredoxin has a disulfide bond, which is in turn reduced by NADPH and thioredoxin reductase. A 6-electron reduction of sulfite produces H₂S, which is incorporated into organic sulfur compounds (mostly cysteine and methionine).

Very important element--essential for amino acid biosynthesis, RNA synthesis, DNA synthesis

Primary acquistion modes:

1. NH₄⁺ uptake (organic or inorganic source)

2. Assimilatory NO₃^- Reduction

3. Nitrogen (N₂ gas) Fixation

Very few reactions in cells use free NH₄⁺, since this is toxic (interferes with pH balance of cytoplasm). Examples include alanine dehydrogenase, glutamate dehydrogenase, glutamine synthetase, and GOGAT (glutamate synthase). Most reactions requiring "NH₄⁺" utilize glutamate or glutamine as NH₄⁺ donors.

Nitrate reduction for assimilation does not produce ATP, but consumes significant energy.

Nitrogenase is very oxygen sensitive and must be protected from oxygen. Very expensive rxn.

Nitrogenase is highly sensitive to oxygen and is rapidly inactivated (irreversibly) by exposure to oxygen. For this reason, cells have adopted many different strategies to protect nitrogenase from inactivation.

Examples: 1. high respiratory activity to maintain low intracellular oxygen concentration.

2. Many bacteria only fix nitrogen when living in anaerobic environments (this may even be the case for facultative anaerobes--only fix nitrogen when growing anaerobically or microaerophilically).

3. Cyanobacteria: fix nitrogen at night when no oxygen evolution can occur from photosynthesis and when respiration maintains low oxygen concentration inside cells (TEMPORAL separation/protection). Other cyanobacteria when grown in nitrogen-limiting conditions differentiate specialized cells known as HETEROCYSTS. This is SPATIAL separation/protection. These cells are quite specialized and are terminally differentiated--cannot divide any longer due to DNA rearrangements). Heterocysts do not have Photosystem II activity and hence do not evolve oxygen. Heterocysts do retain Photosystem I which can provide ATP and low-potential reductant for nitrogen fixation. They obtain fixed carbon (carbohydrate) from adjacent vegetative cells, and oxidize this to provide electrons for nitrogen fixation. Heterocysts export nitrogen compounds to adjacent vegetative cells fulfilling their nitrogen requirements.

Bacteria have structurally different nitrogenases, and at least three classes are known. The most common one contains Fe and Mo (Molybdenum) as part of the essential cofactor; alternative nitrogenases made under Mo-limiting conditions contain vanadium (V) and Fe; still other nitrogenases made when both V and Mo are limiting contain only Fe at the active site. All enzymes contain the metals as a series of Me-S prosthetic groups that are highly reducing in nature and quite oxygen sensitive.

Phosphorus acquisition differs in two fundamental ways from sulfur and nitrogen:

1. Phosphorus is not oxidized or reduced during normal metabolism (although this is still an open question in biology).

2. Phosphorus is assimilated in fueling pathways, due to its central importance in energy metabolism in cells.

Substrate-level Phosphorylation occurs in cytoplasm (soluble enzymes). Phosphorylated intermediate is "activated" by 1. OXIDATION or 2. DEHYDRATION reactions. Resulting "high-energy" phosphorylated intermediate can phosphorylate ADP to form ATP (or equivalent). Example: dehydration of 2-phosphoglyerate to form phosphoenolpyruvate which can phosphorylate ADP yielding ATP and pyruvate in glycolysis.

Initially proposed by Peter Mitchell in 1962. Postulates that protonmotive force can drive cellular transport processes, motility, ATP synthesis. Electron transport reactions create a proton gradient across cytoplasmic membrane; ATP synthase uses proton gradient to drive the synthesis of ATP

Precursor Metabolite Amount (mmole/g cells)

1. Glucose 6-phosphate 205

2. Fructose 6-phosphate 71

3. Triose phosphate 129

4. 3-Phosphoglycerate 1,496

5. Phosphoenolpyruvate 519

6. Pyruvate 2,833

7. Ribose 6-phosphate 898

8. Erythrose 4-phosphate 361

9. Acetyl-CoA 3,748

10. a-Ketoglutarate 1,079

11. Oxaloacetate 1,787

12. ~P (ATP equivalents) 18,485

13. NADH -3,547

14. NADPH 18,225

15. 1-Carbon units [from serine]

16. NH4+ 10,180

17. S-2 233

Only ~P (ATP equivalents) required for assembly and polymerization from Building Blocks.

To convert the 11 precursor metabolites into the 75 building blocks requires energy, reducing power, 1-Carbon units, NH4+ and S-2. However, some NADH is formed (3,547 mmole/g cells; this is equivalent to 7094 mmole ~P/g cells).

Growth on Rich Medium, containing forms of building blocks which can be transported (amino acids, fatty acids, sugars, nucleosides, etc.) saves a lot of energy and ALL reducing power costs (equivalent to a lot more energy!).

However, book (p. 148) says 85% energy savings. This is not quite true, since from glucose, NADH is produced in making building blocks. The overall savings is still substantial--but is only about 75%.

During aerobic growth of E. coli on glucose, about 70% of the glucose is consumed in glycolysis for precursor metabolite synthesis and about 30% of glucose is consumed in pentose phosphate pathway (see Fig. 5, p. 152). Reducing power is produced by both paths. The TCA cycle functions primarily to provide precursors, not to oxidize acetyl units, since reducing power can be converted to ATP equivalents by oxidative phosphorylation (electron transport chain/ATP synthase). PEP carboxylase helps to provide TCA precuror oxaloacetate.

During anaerobic growth of E. coli on glucose, there is less flow through pentose phosphate path, which becomes primarly precursor producer (problem of redox balance). TCA cycle is exclusively biosynthetic function and does not oxidize acetyl units to CO₂; actually consumes reducing power.

E. coli can be grown aerobically on malate or succinate, although growth on this substrate is much slower than on glucose. Malate is a 4-C acid intermediate of the TCA cycle (see p. 154). This means that sugars must be synthesized by reversing glycolysis and pentose-phosphate paths. Energetically expensive processes--4 ATP equivalents required to convert malate to glucose 6-phosphate.

Growth on acetate is even worse: 8 ATP equivalents required to convert acetate to glucose 6-phosphate.

It quickly becomes obvious why E. coli prefers glucose to malate or acetate as carbon/energy source and grows fastest on this sugar.

1. Phototrophs vs. Chemotrophs. Light vs. Chemicals (organic or inorganic) as energy source

2. Lithotrophs vs. Organotrophs. Inorganic vs. Organic electron sources.

3. Autotrophs vs. Heterotrophs. Carbon dioxide vs. complex organics cmpds as carbon source.

However, only 4 major combinations of the six possibilities can occur, since organic molecules serve as energy, carbon, and electron source for some bacteria.

1. Photolithoautotrophs (photoautotrophs): Includes cyanobacteria, purple bacteria, green bacteria, lants (no archaea in this category)

2. Photoorganoheterotrophs. Includes some purple bacteria, green nonsulfur bacteria, heliobacteria, halobacteria (archaea), some protozoa (e.g., Euglena sp.)

3. Chemolithoautotrophs (unique to bacteria and archaea--no eucaryotes in this category). Includes bacteria that oxidize inorganic compounds and use CO₂ as carbon source.

4. Chemoorganoheterophs (most common group) Includes: E. coli, most non-phototrophic bacteria, animals, and fungi)

1. Only substrate-level phosphorylation occurs

2. Organic compounds serve as electron donors and acceptors.

3. No electron transport chain used

4. Compounds fermented: mostly carbohydrates, a few amino acids and organic acids

5. Predominant pathway is glycolysis (Embden-Meyerhof)

1. Occur in the dark (not light driven)

2. ATP generation by chemiosmotic coupling via ATP synthase

3. Usually an inorganic e- acceptor (there are exceptions: e.g., fumarate, dimethyl sulfoxide (DMSO), trimethylamine oxide (TMAO)):

a. oxygen = aerobic respiration

b. NO₃^-, SO₄^-2, S₂O₃^-2, CO₂, Fe⁺³ = anaerobic respiration

4. ATP yields are generally much higher via respiration than via fermentation.

Reduced coenzymes (NADH/NADPH/FADH2/QH₂) are reoxidized by electron transport chain, and a proton gradient is established. ATP synthase uses 3H⁺ to affect the release of 1 ATP from the enzyme.

Glycolysis, TCA cycle, and Pentose-Phosphate pathways can all be used to produce reduced coenzymes for ATP production.

1. Nutrient uptake (active transport).

2. Osmotic (turgor) pressure maintenance.

3. pH maintenance.

4. Motility through turning basal body motors.

5. Reverse electron transport to maintain NAD(P)H levels.

6. Waste/ion excretion by active transport.

7. ATP synthesis via ATP synthase.

The latter reaction is one of the most important in the cell, since it makes up most of the ATP deficit seen from earlier analyses of ATP needs for biosynthesis, polymerization,and assembly.

E. coli can vary the compostion of its quinol oxidases: either cyt bd (low aeration: high affinity for oxygen, but low H⁺/O ratio) or cyt bo₃ (high aeration: lower affinity for oxygen, but higher H⁺/O ratio).

P:O ratio refers to the number of ATPs formed per oxygen atom consumed. This number can vary quite a bit in E. coli, since composition of electron transport chain can vary considerably.

H⁺:P ratio = 3

P:O ratio = 0.75 - 2.0

Some substrates (glycerophosphate, D-lactate, and succinate) send electrons directly to quinone pool rather than NAD(P)H. Composition of the Electron Transport Chain can also vary (menaquinone vs. ubiquinone under anaerobic vs. aerobic conditions).