Chapter 12

Anabolism

• use energy from catabolism

  • carbon source and inorganic molecules → new organelles and cells

• macromolecules are synthesized from monomers

• enzymes do double duty

  • both catabolic and anabolic pathways

    • not identical

• reactions are physically separate

  • compartmentalization

• uses NADPH to donate electrons

• self-assembly

  • macromolecules are assembled into more complex structures

CO2 fixation

• only done by autotrophs

• most used pathways

  • Calvin-Benson (Calvin) cycle

  • Reductive TCA cycle

• other cycles are mostly found in archaea

  • hydroxypropionate bi-cycle

  • reductive acetyl-coa pathway

  • 3-hydroxypropionate/4-hydroxybutyrate

Another name for the Calvin-Benson Cycle

reductive pentose phosphate cycle

Where does the Calvin cycle occur

eukaryotes: stroma of chloroplasts

cyanobacteria, nitrifying bacteria, and thiobacilli: carboxysomes

What is used per one CO2 in the Calvin cycle

3 ATP and 2 NADPH

Why must the Calvin cycle operate 6x

• to produce F6P or G6P

• to produce 12 G3P

  • 2 G3P → glucose

  • 10 G3P → to regenerate (6) RuBP

Carboxylation phase of Calvin cycle

• fixes carbon dioxide

• catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO)

• CO2 + ribulose-1,5-bisphosphate (RuBP) → (2) 3-phosphoglycerate (PGA)

Reduction phase of Calvin cycle

• (2) 3PGA → G3P

  • (2) G3P is used to form glucose

  • NADPH is the reducing power

  • ATP is used

Regeneration phase of Calvin cycle

• (10) G3p is used to regenerate (6) RuBP so that the cycle can repeat

• F6P and G6P are produced as intermediates

• reverse of the PPP

Calvin cycle equation

6CO2 + 18ATP + 12NADPH + 12H + 12H2O → glucose + 18ADP + 18 Pi + 12 NADP+

What uses the Reductive TCA cycle

• chemolithoautotrophs

  • most are strict anaerobes

    • relies on ferredoxin (oxygen-sensitive)

What does the Reductive TCA Cycle need

• fumarate reductase

• alpha-ketoglutarate synthase

• ATP-dependent citrate lyase

Overall reaction of Reductive TCA cycle

4 CO2 → oxaloacetate

• oxaloacetate → PEP

  • with the help of pyruvate and PEP synthases

What is the reverse of EMP

gluconeogenesis

What enzymes do gluconeogenesis use

• Glucose 6-phosphatase

• Fructose bisphosphatase

• PEP carboxykinase

• Pyruvate carboxylase

Synthesis of Sugars

• Uridine diphosphate glucose (UDP-G)

  • G1P + UTP

• UDP-NAM-pentapeptide and UDP-NAG

  • peptidoglycan synthesis

What does the synthesis of amino acids need

• multiple methods and steps contribute needed

  • nitrogen assimilation

  • sulfur assimilation

    • for cysteine and methionine

  • amino acid biosynthetic pathways

  • anaplerotic reactions and amino acid biosynthesis

Assimilatory pathways

• inorganic nitrogen and sulfur are incorporated into organic materials

  • protein, nucleic acids, coenzymes, and other cell constituents

Dissimilatory pathways

• inorganic compounds are used instead of oxygen as electron acceptors

  • such as in anaerobic respiration

    • P. denitrificans and E. coli

  • product is excreted into the environment

What are potential sources of nitrogen

• ammonia (NH3, more reduced)

• nitrogen (N2)

• nitrate (NO3, more oxidized)

Ammonia Oxidizing Bacteria (AOB)

• chemoautotrophic

  • only uses ammonia for energy

• ammonia → nitrate

Ammonia Oxidizing Archaea

• ammonia → nitrite

• lower pH than AOB

Nitrite Oxidizing Bacteria

nitrite into nitrate

Anammox

ammonia + nitrite → nitrogen gas

Assimilatory Nitrate Reduction

• used by bacteria to reduce nitrate → ammonia

• occurs in the cytoplasm of bacteria

  • nitrate → nitrite via nitrate reductase

    • NO3- → NO2-

  • nitrite → ammonia (NH3) via nitrite reductase

What is nitrogen fixation

atmospheric gaseous nitrogen → NH3

• catalyzed by nitrogenase

  • highly sensitive to oxygen

What carries out nitrogen fixation

• a few prokaryotes (diazotrophs)

  • chemotrophic bacteria and archaea (Klebsiella)

  • plant symbionts (Rhizobium)

  • cyanobacteria

What does nitrogen fixation need

8 electrons and 16 ATP molecules

• it is exergonic

Ammonia Incorporation

• Reductive amination

  • when [NH3] is high, no ATP needed

    • still uses NADPH as reducing power

  • alpha-ketoglutarate → glutamate

    • via glutamate dehydrogenase (GDH)

• Glutamine synthetase-glutamate synthase (GS-GOGAT)

  • when [NH3] is low, uses ATP

  • glutamate → glutamine

    • via glutamine synthetase

  • alpha-ketoglutarate + glutamine → (2) glutamate

    • via glutamate synthase

What is sulfur needed for

• synthesis of amino acids

  • methionine and cysteine

• synthesis of coenzymes

  • CoA

Where is sulfur obtained from

• external sources

• intracellular amino acid reserves

Assimilatory sulfate reduction

• sulfate (SO2-4), more oxidized form

• phosphoadenosine 5’-phosphosulfate (PAPS)

  • activates sulfate so that it can be reduced

• sulfate reduced to H2S → cysteine

Amino acid biosynthesis

• regulated by feedback mechanisms

• single precursor → several amino acids

  • branching pathways

  • oxaloacetate → lysine, threonine, isoleucine, and methionine

  • alanine is made from pyruvate

  • aspartate is made from oxaloacetate

Aromatic amino acid biosynthesis

tryptophan, phenylalanine, and tyrosine from chorismate (E4P + PEP)

Purines and Pyrimidines synthesis

• most microbes can synthesize their own purines and pyrimidines

  • needed to make ATP, RNA, and DNA

• phosphorous assimilation is needed

Phosphorus Assimilation

• assimilation occurs during fueling

• most common phosphorus sources are:

  • inorganic phosphate (Pi)

    • incorporate through the formation of ATP by

      • photophosphorylation

      • OP

      • SLP

  • organic phosphoryl group

    • present in environment in dissolved or particulate form

    • hydrolyzed by phosphatases, releasing Pi

      • enzymes can be found in the periplasmic space of Gram-negative

Purine biosynthesis

• seven different molecules contribute

  • begins with ribose 5-phosphate

  • folic acid cofactor

  • uses aspartate, glutamine, glycine, and CO2

• initial products are ribonucleotides

  • inosinic acid → AMP and GMP

  • not a free purine base

    • built directly onto the sugar

• deoxyribonucleotides formed by reduction

  • requires thioredoxin (sulfur-containing protein)

  • via vitamin B12 as a cofactor

Pyrimidine Biosynthesis

• exists as free bases

  • construction is completed before adding ribose

• aspartic acid + bicarbonate and glutamine

• deoxyribose forms of U and C nucleotides formed by reduction of ribose

  • same as purines (thioredoxin and Vitamin B12)

  • U → T via methylation by a folic acid derivative

Lipid Synthesis

• Component of cell membranes

  • also outer membrane of Gram-negatives (LPS)

• bacteria and eukaryotes: fatty acid or derivatives

  • ester linkage

• archaea: isoprene

  • ether linkage

  • monolayer or bilayer

Fatty Acid Synthesis

• Saturated fatty acids by fatty acid synthase complex

  • from acetyl-CoA, malonyl-CoA, and NADPH

  • two carbons added at a time

• Unsaturated FA by added double bond

  • eukaryotes and aerobic bacteria: NADPH + O2 → H2O

  • anaerobic bacteria and some aerobes: dehydration of hydroxyl fatty acids

• branched pathway to make triacylglycerol and phospholipids

Isoprene Lipid Synthesis

• synthesized by pathways that start with acetyl-CoA

• archaeal building blocks are intermediates of the sterol pathway:

  • isopentenyl pyrophosphate (IPP)

  • dimethylallyl pyrophosphate (DMAPP)

Lipopolysaccharides (LPS) Synthesis

• Gram-negative bacteria

  • lipid A, oligosaccharide core, O-polysaccharide (O-antigen), KDO

• LPA has two branches

  • lipid A attachment to core (lipid A-core)

    • starts with UDP-NAG, includes addition of KDO

    • synthesized in the cytoplasm

  • O antigen repeat unit and polymerization

    • added to bactoprenol in the periplasm

• cell membrane to outer membrane

  • by Lpt proteins

Precursor Metabolites

• generation of precurose metabolites is a critical step in anabolism

  • E4P - aromatics: phenylalanine, tyrosine, and tryptophan

  • R5P - nucleosides

  • G3P - lipids

  • Acetyl Co-A - lipids

  • Oxaloacetate - aspartate

What is the goal for the Calvin cycle

3 CO2 → G3P

Purines

• double ring

• adenine, guanine

Pyrimidines

• single ring

• cytosine, thymine, and uracile

What are involved in purine synthesis

aspartate/aspartic acid, glycine, glutamine, folic acid, R5P, and CO2

What amino acid is involved in pyrimidine synthesis

aspartic acid

Cysteine biosynthesis

• fungi: H2S + Serine → Cysteine

• bacteria + archaea: Serine → O-acetylserine → Cysteine

• archaea: 3PG →→ O-phosphoserine → cysteine