Mayer

Anabolism: the building of complex biomolecules

• amino acids/proteins

• Carbohydrates/polysaccharides/peptidoglycan

• Nucleotides/RNA/DNA

• Fatty acids, polyketides

• Other biomolecules/ secondary metabolites/non ribosomal peptide synthesis

• essential elements: C H N O P S & other essential elements

• Reduction: by reducing agents such as NADPH

• Energy: by coupling reactions to ATP hydrolysis NADPH oxidation or iron flow down a trnasmembrane concentration gradient

• Many substrates for biosynthesis arise from gylcolysis/gluconeogenesis & the TCA cycle

• transcriptional/translational regulation, allosteric

• Competition/predation: antibiotics, toxins

• Genome degradation: reductive evolution (flagella, motility)

• consists of samll (S) & large (L) subunits

• Catalyzes the condensation of CO2 to ribulose 1,5 bisphosphate & the splitting of the unstable 6C intermediate into two 3C 3-phosphgylcerate molecules

1. carboxylation & splitting of 6C -> 2[3C]: ribulose-1,5-bisphosphate condenses with CO2 & H2O to form a 6C molecule ehich immediatly splits into two 3-phosphoglycerate molecules

2. Reduction of PGA to Glyceraldehyde 3-P: the carboxlygroup of PGA is phosphorylated by ATP & then hydrolyzed & reduced by NADPH, this generates G3P

3. Regeneration of ribulose 1,5-bisphosphate: one of every six G3P is converted to glucose, the other five molecules enter a series of reactions that regenerate three molecules of ribulose 1,5-bisphosphate

Many organisms contain the Rubisco complex within subcellular structures called carboxysomes

-> the carboxysomes take up bicarbonate (HCO3-) which is then immediatly converted to CO2 by carbonic anhydrase, the CO2 is then fixed by Rubisco

Regeneration steps to regenerate TCA cycle intermediates out of small amounts of CO2

E.g. phosphoenolpyruvate + Co2 -> oxaloacetate (phosphoenolpyruvatcarboxylase)

• in some bacteria & archaea the entire TCA cycle functions in “reverse”: allows the reduction of CO2 to regenerate Acetyl-CoA & build sugars

• Uses 4-5 ATPs to fix four molecules of CO2 & generate one oxaloacetate

• Reduction (addition of 2H+ + 2e-) is performed by NADPH & NADH & by reduced ferredoxin (FDH2)

• reductive Acetyl-CoA pathway

• Used by anaerobic soil bacteria, autotrophic sulfate reducers & methanogens

• Two CO2 molecules are condensed through converging pathways to form the acetyl group of Acteyl-CoA - Carbonmonoxid as an intermediate

• Reducing agent is H2 instead of NADPH

• used by thermophiles such as the bacterium chloroflexus & the archaea sulfolobus

• Acetyl-CoA condenses with hydrated CO2 (bicarbonate ion HCO3-) & is reduced by 2 NADPH to 3-hydroxypropionate

• Intermediates includes methylmalonyl-CoA

• In all three molecules of CO2 are into one molecule of pyruvate which serves as substrate for biosynthesis

• the cyclic process of fatty acid synthesis is managed by the fatty acid synthase complex

• Molecules of acetyl-CoA are carboxylated to Malonyl-CoA

• To the coenzymeA is replaced by acyl-carrier-protein (ACP) making malonyl-ACP

• Malonyl-ACP condenses with the growing (ACP-) chain

• The growing chain now contains a ketone which us reduced to CH2 by 2 NADPH

• Successive addition con continue many times to build a saturated fatty acid of indefinite length

• Acetyl-CoA -> Acetyl-ACP only used as primer

• Unsaturation can be generated during a cycle of elongation: a specila dehydratatse enzyme forms the double bond between the third and fourth carbons

• Trans double bonds would interfere with membrane: cis double bonds needed

• fatty acid synthesis consumes large quantities of reducing energy & so must be regulates closely

• In E. Coli key points of regluation include:

  • Acetyl-CoA carboxylase regulates its own transcription

  • Starvation blocks fatty acid synthesis through the “stringent response”

  • Low temperature favors unsaturated fatty acids by inducing expression of the dehydratase enzyme

• polyketides are a diverse group of metabolites that include the broad-spectrum antibiotic erythromycin

• Polyketides are snythesized by an enormous enzyme complex called a modular enzyme: consits of multiple modules which add smilar but non-identical units to a growing chain

• Like fatty acids polyketides are built by the successive condensaion of malonyl-ACP units: but each malonyl group carries a unique extension or R-group; these are thyl groups in the case of erythromycin

B: legume symbionts

• nitrogen gas (N2) is fixed into ammonium ions (NH4+) only by some species of bacteria and archaea

• Aquatic cyanobacteria developed special cells (heterocysts) to fix N2: photosynthesis is turned off to maintain anaerobic conditions, in the presence of oxygen iron sulfur clusters of nitrogenase would be destroyed

• in living cells nitrogen fixation is an enormously nenergy intensive process

• The mechanism is largely conserved across species

N2 + 8H+ + 8e- +16ATP -> 2NH3 + H2 + 16ADP + 16Pi

• about 40 ATPs are consumed per N2 fixed (including intermediates)

• Nitrogen fixation is catabolyed by the enzyme Nitrogenase

• the nitrogenase complex includes two kinds of subunits

  • a protein with an iron-sulfur core (Fe protein)

  • A protein containing a complex of molybdenum, iron & sulfur proteins (FeMo-protein)

• Electrons acquired by Fep protein (with energy from ATP) are transferred to FeMo protein to reduce nitrogen

• Nitrogen fixation requires four reduction cycles through nitrogenase

1. Fe protein acquires 2e- from an electron transport protein such as ferredoxin & then trnasfers them to the FeMo center

2. The FeMo center binds 2H+ which is reduced to H2 gas

3. N2 can now bind the active site by displacinds the H2

4. Successive pairs of H+ & e- reduce

N2 -> HN=NH -> H2N-NH2 -> 2NH3

• nitrogen fixation costs substantial energy & therfore the process is highly regulated

• Oxygen & NH4+ availability regulate expression of the nif genes which encode nitrogenase & other nitrogen fixation proteins

• Regulation of nif is mediated by several molecular regulator including

  • a nitrogen starvation sigma factor (sigma54)

  • NtrB-NtrC two component signal trasnduction system

• The carbon skeletons of amino acids arise from diverse intermediates of metabolism

• Certain amino acids reise directly from key metabolic intermediates: e.g. glutamate from 2-oxoglutarate

• Other amino acids must be synthesizes from preformes amino acids: e.g. glutamine & arginine from glutamate

• Still other amino acids can arise from more than one source: e.g. leucine & isoleucine can be made from succinate as well as pyruvate

• most bacteria assimilate NH4+ by condensing it with 2-oxogluterate to form glutamate or with glutamate to form glutamine

• Both glutamate & glutamine contribute an amine as well as their C skeleton to the synthesis of other amino acids in the biosynthesis

• The transfer of ammonia between the two metabolites as called

  • e.g. glutamate transfers NH3 to oxaloacetate making aspartet & 2 oxogluterate, this reaction can also be reverses

• The cellular levels of glutamate & glutamine act as indicators of nitrogen availability

• Some amino acids require longer pathways involving numerous enzymes

• E.g.

  • arginine biosynthesis generally involves about a dozen different enzymes distributing among four to eight operons

  • Aromatic amino acids are built from a common pathway that branches out

• Synthesis is tightly regulated at both transcriptional & translational levels

• both purines & pyrimidines are built onto a ribose-5-phosphate substrate, converted into 5-phosporibosyl-1-PP via the activation step of ATP -> AMP

• The purine ring is built out of succcessive additions of amines plus a formyl group

  • First purine made is inosine

  • Then converted to AMP & GMP

• Pyrimidines are built by a slightly different rout

  • first pyrimidine made is uracil

  • Then converted to CMP & TMP