Ch. 12: Anabolism - The Use of Energy in Biosynthesis

anaplerotic reaction: balancing energy (ATP) and AA/molecules

anabolism uses energy from

catabolism

• used for biosynthetic pathways

• carbon source and inorganic molecules → new organelles and cells

macromolecules are synthesized from

monomers

do many enzymes work in both directions?

yes — double duty, but the catabolic and anabolic pathways are not identical

Are anabolic and catabolic reactions physically separated?

Yes, in eukaryotes, these reactions happen in different compartments of the cell (e.g., cytoplasm vs. mitochondria) to prevent interference.

what is the difference between NADH and NADPH in metabolism

NADH - catabolic (like respiration)

NADPH - anabolic

what is a critical step in anabolism

generation of precursor metabolites

what are carbon skeletons used for

amino acids

many prokaryotes can grow in what compounds

C1

ex: CO2, CH4, methanol (CH3OH)

what are the most used CO2 fixation pathways?

Calvin-Benson (calvin) cycle

reductive TCA cycle

what domain are other cycles mostly found in

Archaea

Calvin-Benson cycle

• aka reductive pentose phosphate cycle

• consists of 3 phases

• goal is to make glyceraldehyde 3-P from 3 CO2

• 3 ATPs and 2 NADPHs are used per 1 CO2

Calvin-Benson cycle location

• in eukaryotes: in stroma of chloroplasts

• in cyanobacteria/some nitrifiying bacteria, and thiobacilli: in carboxysomes

carboxysomes

inclusions that usually have a single membrane around them

what are the 3 phases of Calvin-Benson cycle

• the carboxylation phase

• the reduction phase

• the regeneration phase

carboxylation phase

• catalyzed by enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO)

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

reduction and regeneration phases

• RuBP is regenerated

• fructose and glucose are produced

• ATP + NADPH convert 3-phosphoglycerate → glyceraldehyde 3-P

reductive TCA cycle

• used by some chemolithoautotrophs

  • most are strict anaerobes

  • non-fermenter

• reverse of oxidative TCA cycle

• Overall reaction: 4 CO2 → oxaloacetate

  • → Pyruvate → PEP via PEP synthases

what are the 3 things the reductive TCA cycle needs

• fumarate reductase

• α-ketoglutarate synthase 

• ATP-dependent citrate lyase

gluconeogenesis

functional reversal of glycolysis

• the 2 pathways are not identical

enzymes involved in gluconeogenesis

• Pyruvate carboxylase

• Phosphoenolpyruvate carboxykinase

• Fructose bisphosphatase

• Glucose 6-phosphatase

synthesis of sugars involves

• Uridine diphosphate glucose (UDP-G)

  • G1P + UTP

• UDP-NAM-pentapeptide and UDP-NAG

  • PDG synthesis

synthesis of amino acids

• precursor metabolites are used for synthesis of AA

  • amino group and sometimes sulfur are added

• involves multiple methods and steps

  • nitrogen assimilation

  • sulfur assimilation

    • for cysteine and methionine

  • amino acid biosynthetic pathways

  • anaplerotic reactions and AA biosynthesis

anaplerotic reactions

replenish intermediates

balancing energy (ATP) and AA/molecules

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

  • product is excreted into the environment

nitrogen assimilation

• CHONPS!!!

• potential sources of nitrogen

  • ammonia (NH3, most reduced)

  • nitrogen (N2)

  • nitrate (NO3, most oxidized)

ammonia-oxidizing bacteria (AOB)

ammonia (NH₃) → nitrite (NO₂⁻)

Ammonia-oxidizing archaea (AOA)

ammonia (NH₃) → nitrite (NO₂⁻)

• like AOB, but works better in low pH (acidic)

Nitrite-oxidizing bacteria (NOB)

(NO₂⁻) → nitrate (NO₃⁻)

anammox

ammonia + nitrite → nitrogen gas

anaerobic

BOD (biochemical oxygen demand)

• BOD + nitrite/nitrate → nitrogen gas

• denitrification

ammonia → proteins, oxidized nitrogen

in this process, nitrogen goes from reduced to more oxidized state

as microbes use ammonia for energy…

they have reducing power to reduce nitrite

assimilatory nitrate reduction

• used by bacteria to reduce nitrate to ammonia

• occurs in bacteria cytoplasm

  • Nitrate (NO3-) reduction to nitrite (NO2-) catalyzed by nitrate reductase

  • Nitrite reduction to ammonia (NH3) catalyzed by nitrite reductase

• req nitrate reductase, nitrite reductase, and NADPH

nitrogen fixation

• Reduction of atmospheric gaseous nitrogen to NH3 

  • catalyzed by enzyme nitrogenase

    • highly sensitive to oxygen

• only carried out by few prokaryotes (diazotrophs)

  • Chemotrophic bacteria and archaea (Klebsiella)

  • Plant symbionts (Rhizobium)

  • Cyanobacteria

• Process is exergonic

  • needs 8 electrons and 16 ATP molecules

  • nitrogen fixing bacteria can use up to 20% of the ATP made by the host plant!

2 processes that incorporate ammonia

• reductive amination

• Glutamine synthetase-glutamate synthase (GS-GOGAT)

Reductive amination

• when [NH3] is high, no ATP needed

• α-ketoglutarate → glutamate by glutamate dehydrogenase (GDH)

Glutamine synthetase-glutamate synthase (GS-GOGAT)

• when [NH3] is low, uses ATP

• glutamate → glutamine by glutamine synthetase

• α-ketoglutarate + glutamine → glutamate by glutamate synthase

sulfur assimilation

• Sulfur is needed for

  • synthesis of amino acids

    • Methionine and cysteine

  • synthesis of several coenzymes

• Sulfur obtained from

  • external sources 

  • intracellular amino acid reserves

• Assimilatory sulfate reduction

  • sulfate (SO^2-₄, inorganic), more oxidized form

  • phosphoadenosine 5’-phosphosulfate (PAPS)

    • intermediate before sulfate is reduced to sulfite

  • sulfate reduced to H2S → cysteine

    • (a) fungi, (b) bacteria and archaea, (c) archaea (see pic)

Amino Acid Biosynthesis

• Regulated by feedback mechanisms

• Single precursor → several amino acids

  • this is a branching pathway

  • ex: oxaloacetate → lysine, threonine, isoleucine, and methionine

• Aromatic amino acids

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

Purines and Pyrimidines Synthesis

• Most microbes can synthesize their own purines and pyrimidines which allows for the use of AA

• Amino acids participate in this process

  • pyrimidine (C/T/U) - made by aspartic acid

  • purine (A/G) - made by aspartic acid, glycine, glutamine

• Phosphorous assimilation is needed

Phosphorous Assimilation

• Assimilation occurs during fueling

• Most common phosphorus sources are:

  • Inorganic phosphate (Pi) 

  • Organic phosphoryl group

inorganic phosphate (Pi)

• incorporated through the formation of ATP by

  • Photophosphorylation

  • oxidative phosphorylation (OP)

  • substrate-level phosphorylation (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

• 2 rings, adenine and guanine

• Seven different molecules contribute

  • begins with ribose 5-phosphate 

  • folic acid cofactor

• Initial products are ribonucleotides

• Deoxyribonucleotides formed by reduction

  • requires thioredoxin (sulfur-containing protein)

  • via vitamin B12 as a cofactor

• Aspartate and glycine

pyrimidine biosynthesis

• 1 ring, thymine and cytosine

• 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

  • U → T via methylation by a folic acid derivative

• Aspartic acid and glutamine

lipid synthesis

• Component of cell membranes

  • also outer membrane of Gram-negatives

• Bacteria and eukaryotes: fatty acid or derivatives

• Archaea: isoprene

fatty acid (FA) synthesis

• Saturated FA made 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: double bond added by NADPH + O2 → H2O 

  • anaerobic bacteria and some aerobes: double bond added by dehydration of hydroxyl fatty acids 

• Branched pathway that also makes triacylglycerol and phospholipids

isoprene lipids

• Isoprene commonly synthetized by pathway that starts with acetyl-CoA

• Archaeal building blocks are intermediates of the sterol pathway:

  • isopentenyl pyrophosphate (IPP) 

  • dimethylallyl pyrophosphate (DMAPP)

lipopolysaccharides (LPS)

• found in gram-negative bacteria

• LPS biosynthesis has two branches:

  • lipid A-core synthesis

    • starts with UDP-NAG, includes addition of KDO

  • O antigen synthesis

    • O-antigen is made as repeat sugar units that are polymerized

    • added to bactoprenol (shuttle across membrane)

• LPS moves from cell membrane to outer membrane

  • by Lpt proteins

LPS structure

• lipid A

• oligosaccharide core

• O-polysaccharide (O antigen)