Fundamentals in Biology 1 PDF

Fundamentals in Biology 1, J. Piel Fundamentals in Biology 1: Essentials of Anabolism Introduction to anabolism Relationship to catabolism How do elements enter cells? Carbon fixation: Example Calvin-Benson-Bassham cycle Nitrogen fixation How do cells build bi...

Fundamentals in Biology 1, J. Piel Fundamentals in Biology 1: Essentials of Anabolism Introduction to anabolism Relationship to catabolism How do elements enter cells? Carbon fixation: Example Calvin-Benson-Bassham cycle Nitrogen fixation How do cells build biomolecules? Carbohydrate biosynthesis: gluconeogenesis and pentose phosphate pathway Fatty acid biosynthesis Amino acid biosynthesis: nitrogen incorporation and biosynthetic principles Nucleotide biosynthesis Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Overview anabolism Anabolism is the sum of cellular pathways that build more complex biomolecules from simpler precursors. Usually endergonic and powered by high-energy molecules generated by catabolism. One common principle is activation of biomolecules by ATP or other nucleotide triphosphates to increase the reactivity, like in this example on peptide and protein biosynthesis: AMP + PPi Anabolism (constructing) … Peptides and Purpose: proteins build the cell ATP High-energy See J. Vorholt’s part ATP tRNA on metabolic coupling and N. Ban’s part on -PPi -AMP protein biosynthesis Low-energy High-energy High-energy, ready for protein biosynthesis Amino acid connection by water removal is energetically not feasible. Amino acids are first converted to a high- energy compound with a reactive bond. Coupling the process to ATP “hydrolysis” makes the reaction exergonic. Note: “Hydrolysis” refers to the end products of ATP, but ATP is not actually hydrolyzed. Placeholder for organisational unit name / logo (edit in slide master via “View” > “Slide Master”) | |  Fundamentals in Biology 1, J. Piel Overview anabolism Anabolism is the sum of cellular pathways that build more complex biomolecules from simpler precursors. Usually endergonic and powered by high-energy molecules generated by catabolism. One common principle is activation of biomolecules by ATP or other nucleotide triphosphates to increase the reactivity, like in this example on peptide and protein biosynthesis: AMP + PPi Anabolism (constructing) … Peptides and Purpose: proteins build the cell ATP Catabolism (deconstructing) Nutrients: Purpose: CO2, waste products Sugars, lipids etc. create chemical energy ATP generated by catabolism (glycolysis, the TCA cycle, oxidative phosphorylation) makes these bond connections possible. Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Overview anabolism In addition, many complex biomolecules are reduced compared to their precursors and require reduced coenzymes for biosynthesis, such as NADPH and FADH2, that are generated from catabolic processes. Fatty acid biosynthesis: 2 NADP+ + CO2 Anabolism (constructing) Fatty Purpose: acids build the cell Enz = enzyme 2 NADPH + 2H+ Catabolism (deconstructing) Nutrients: Purpose: CO2, waste products Sugars, lipids etc. create chemical energy Real process is more complex. More details on fatty acid biosynthesis on Thursday. Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Similar core pathways are used in catabolism and anabolism Catabolism (deconstructing, “energy-creating”) Anabolism (constructing) Sugars Sugars Nucleotides Glycolysis Glycolysis/ Gluconeogenesis ATP, Amino NADH acids Fatty Fatty Acetyl-CoA Acetyl-CoA acids acids TCA NAD(P)H, TCA cycle FADH2 cycle Oxidative phosphorylation ATP CO2 ATP Input: central metabolites, ATP, NADPH, Placeholder for organisational unit name / logo (edit in slide master via “View” > “Slide Master”) FADH2, etc. | |  Fundamentals in Biology 1, J. Piel Anabolism is complex… Ø Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”) https://web.expasy.org/pathways/  Fundamentals in Biology 1, J. Piel This lecture: CO2 and N2 fixation (= assimilation) Sugars Nucleotides Glycolysis/ Gluconeogenesis Carbon fixation Nitrogen fixation Amino CO2 acids N2 Fatty Acetyl-CoA acids Organisms that can grow on CO2 as Nitrogen fixers are called the sole carbon source are termed TCA diazotrophs. Nitrogen fixation is not a autotrophs (primary producers); cycle defining feature of autotrophy; many those that cannot are heterotrophs. nitrogen-fixing organisms are heterotrophs, i.e., they rely on organic carbon. Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Major players in CO2 fixation Important marine groups Cyanobacteria The cyanobacterial genus Prochlorococcus is among the wikimedia commons most abundant photosynthetic groups of organisms on earth (but was discovered only in 1986). *large error margin Sources: Proc. Natl. Acad. Sci. U. S. A. 2016, E5354 Proc. Natl. Acad. Sci. U. S. A. 2012, 109: 8845 Annu. Rev. Mar. Sci. 2011, 3:291 Plant Physiol. 2004, 135:2106 Sallie (Penny) Chisholm, Placeholder for organisational unit name / logo MIT | | (edit in slide master via “View” > “Slide Master”) S. W. Chisholm et al., Nature. (1988) 334:340 https://www.sciencemag.org/news/2017/03/meet-obscure-microbe-influences-climate-ocean-ecosystems-and-perhaps-even-evolution  Fundamentals in Biology 1, J. Piel Major players in CO2 fixation Important marine groups Cyanobacteria The cyanobacterial genus Prochlorococcus is among the wikimedia commons most abundant photosynthetic groups of organisms on earth (but was discovered only in 1986). Protists (eukaryotic) Dinoflagellates Coccolithophores wikimedia commons *large error margin Sources: Proc. Natl. Acad. Sci. U. S. A. 2016, E5354 Proc. Natl. Acad. Sci. U. S. A. 2012, 109: 8845 Diatoms wikimedia commons Annu. Rev. Mar. Sci. 2011, 3:291 wikimedia commons Plant Physiol. 2004, 135:2106 Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Autotrophy in cyanobacteria and plants at the molecular level Net reactions (oversimplified): Oxidation 6 CO2 + 6 H2O C6H12O6 + 6 O2 Storage granules J. Bacteriol. 1983, 393; wikimedia commons Reduction Photosynthesis Energy + 6 CO2 + 6 H2O C6H12O6 + 6 O2 Cyanobacterial glycogen Respiration Morris et al. How life works 2e, Freeman & Co., Fig. 8.2 C6H12O6 + 6 O2 6 CO2 + 6 H2O + Energy More precise: Potato starch In microorganisms and plants, glucose and other sugars are mainly produced as phosphates or incorporated into polymeric storage carbohydrates (glycogen in cyanobacteria, starch in plants). Many Placeholdercompounds other for organisational unit name / logo than carbohydrates are made. | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel The Calvin-Benson-Bassham (CBB) cycle is the most important process of CO2 fixation Other names: Calvin cycle, Calvin-Benson cycle wikimedia commons Shutterstock Shutterstock Melvin Calvin Green alga Chlorella vulgaris 1961 Nobel Prize in Chemistry Various sugars and CO2 other biomolecules Converts >90% of Earth’s inorganic carbon to biomass (including yours). Estimations for annual Placeholder for organisational unit name / logo (edit in slide master via “View” > “Slide Master”) amount of CO2 fixed via the CBB cycle: 2-4 x 1011 tons (gigatons)! | |  Fundamentals in Biology 1, J. Piel The CBB cycle is the predominant mechanism of CO2 fixation wikimedia commons Shutterstock pxhere.com Cyanobacteria Algae Plants Fuchs, Allg. Mikrobiologie, Thieme, microbewiki.Kenyon.edu wikimedia commons Fig. 11.6 Purple bacteria Nitrifying bacteria (consume oxygen, electrons from (chemolithotrophs) Placeholder for organisational unit name / logo (edit in slide master via “View” > “Slide Master”) anoxygenic photosynthesis) | |  Fundamentals in Biology 1, J. Piel Calvin-Benson-Bassham cycle: Overview Overall stoichiometry: Input CO2 ATP 6 CO2 + 12 NADPH + 18 ATP ADP C6H12O6(PO3H2) + 12 NADP+ + 18 ADP + 17 Pi* *16 Pi from CBB cycle and one from conversion of GAP to glucose 6-phosphate Carboxylation Purpose: Biomass generation Reduction Accomplish the incorporation of CO2 to generate ADP NADPH an organic molecule, 3-phosphoglycerate, the primary product of fixation, and conversion to the ATP NADP+ central metabolite glyceraldehyde 3-phosphate Pi Regeneration of (GAP) so that synthesis of carbohydrates and the carboxylation other metabolites becomes feasible. substrate Generate additional four-, five-, six-, and seven- carbon compounds from three-carbon compounds (see later). Pi Output The CBB cycle has to run six times to create Sugars and other Glyceraldehyde 3- one molecule of glucose 6-phosphate, a hexose biomolecules phosphate (GAP) Placeholder for organisational unit name / logo (= (edit in6-carbon) sugar. slide master via “View” > “Slide Master”) | |  Fundamentals in Biology 1, J. Piel Calvin-Benson-Bassham cycle Overall stoichiometry: 6 CO2 12 ATP 6 CO2 + 12 NADPH + 18 ATP 12 C3 12 ADP C6H12O6(PO3H2) + 12 NADP+ + 18 ADP + 17 Pi* *16 Pi from CBB cycle and one from conversion of GAP to glucose 6-phosphate Purpose: 6 C5 12 C3 Biomass generation Carbon chain lengths change Accomplish the incorporation of CO2 to generate 6 ADP twice. Note the preserved an organic molecule, 3-phosphoglycerate, the overall carbon numbers 12 NADPH primary product of fixation, and conversion to the (adjusted to 1 molecule of 6 ATP 12 NADP+ glucose-6-phosphate as the central metabolite glyceraldehyde 3-phosphate ultimate product) 12 Pi (GAP) so that synthesis of carbohydrates and other metabolites becomes feasible. Glyceraldehyde 6 C5 12 C3 3-phosphate Generate additional four-, five-, six-, and seven- (GAP) carbon compounds from three-carbon GAP compounds (see later). 10 C3 4 Pi The CBB cycle has to run six times to create Sugars and other one molecule of glucose 6-phosphate, a hexose C6 2 C3 GAP biomolecules Placeholder for organisational unit name / logo (= (edit in6-carbon) sugar. slide master via “View” > “Slide Master”) | |  Fundamentals in Biology 1, J. Piel Calvin-Benson-Bassham cycle Overall stoichiometry: 6 CO2 12 ATP 6 CO2 + 12 NADPH + 18 ATP 12 12 ADP C6H12O6(PO3H2) + 12 NADP+ + 18 ADP + 17 Pi* P P *16 Pi from CBB cycle and one from P conversion of GAP to glucose 6-phosphate Purpose: 6 12 Biomass generation Carbon chain lengths change P Accomplish the incorporation of CO2 to generate 6 ADP P twice. Note the preserved an organic molecule, 3-phosphoglycerate, the overall carbon numbers 12 NADPH primary product of fixation, and conversion to the (adjusted to 1 molecule of 6 ATP 12 NADP+ glucose-6-phosphate as the central metabolite glyceraldehyde 3-phosphate ultimate product) 12 Pi (GAP) so that synthesis of carbohydrates and other metabolites becomes feasible. 6 Glyceraldehyde 12 3-phosphate Generate additional four-, five-, six-, and seven- GAP (GAP) carbon compounds from three-carbon P P compounds (see later). 10 4 Pi P The CBB cycle has to run six times to create Sugars and other one molecule of glucose 6-phosphate, a hexose biomolecules P 2 GAP Placeholder for organisational unit name / logo (= (edit in6-carbon) sugar. slide master via “View” > “Slide Master”) P | |  Fundamentals in Biology 1, J. Piel The CO2 Fixation Step 3-Phospho- glycerate Incorporation of CO2 into the organic molecule 6 CO2 ʘ 12 ATP ribulose 1,5-bisphosphate by the enzyme ribulose 12 1,5-bisphosphate carboxylase (RuBisCO) 12 ADP 6 RuBisCO P ʘ 12 CO2 + Ribulose 1,5- P 6 ADP bisphosphate 12 NADPH Ribulose 1,5- Unstable intermediate 6 ATP bisphosphate 12 NADP+ H2O 12 Pi 6 Glyceraldehyde 12 3-phosphate GAP (GAP) Product: P P 3-Phosphoglycerate 10 (2 equivalents) 4 Pi P Sugars and other biomolecules P 2 GAP Placeholder for organisational unit name / logo (edit in slide master via “View” > “Slide Master”) P | |  Fundamentals in Biology 1, J. Piel Ribulose-1,5-bisphosphate carboxylase (RuBisCO) Other spellings: Rubisco, RuBisCo, RubisCO RuBisCo is an unusually slow enzyme and fixes only about three CO2 molecules per second. This is compensated by very high enzyme concentrations. RuBisCO is likely the most abundant enzyme on earth! It is synthesized at about 4 x 109 tons per www.rcsb.org/structure/9rub year and fixes 1011 tons of CO2 per year. proteopedia.org Let’s have a closer look at the enzyme to understand why RuBisCO is so inefficient. RuBisCO from the bacterium Spinach RuBisCO Rhodospirillum rubrum The simplest forms of RuBisCO consisting of two subunits exist in archaea and bacteria (left, one active site encircled). Plant enzymes are more complex (right, Placeholder for organisational unit name / logo (edit in8 slideactive sites> “Slidein red). | | master via “View” Master”) Proc. Natl. Acad. Sci. U. S. A. 2019, 116:4738  Fundamentals in Biology 1, J. Piel RuBisCO is slow... and faulty Challenge 1: CO2 is a small, linear, featureless molecule that is difficult to bind. The Mg2+ ion helps to Ø polarize it and bring it close to the substrate ribulose- 1,5-bisphosphate. Ø Stryer Biochemistry 8e, Macmillan, Fig. 20.3 CO2 Mg2+ www.wikiwand.com/en/RuBisCO Challenge 2: Oxygen (O2) resembles CO2 in its small size, absent molecule dipole, and linear structure. It also fits into the binding site to result in a wasteful side reaction. Active site of RuBisCO from the red alga Galdieria sulphuraria with CO2 bound. The yellow arrow points to a Mg2+ ion that is important for substrate binding. Placeholder for organisational unit name / logo CO2 O2 | | (edit in slide master via “View” > “Slide Master”) Proc. Natl. Acad. Sci. U. S. A. 2012, 109:18785  Fundamentals in Biology 1, J. Piel Photorespiration, a wasteful side reaction of RuBisCO using O2 Modified from Stryer Biochemistry 8e, Macmillan, Fig. 20.3 Oxygen instead of CO2 attacks ribulose-1,5-bisphosphate, intermediate falls apart: ʘ Toxic, has to be Phosphoglycolate recycled by additional enzymes 3-Phosphoglycerate Ribulose 1,5- Unstable hydroperoxide bisphosphate intermediate Photorespiration wastes 25-30% of the photosynthetic energy in plants. Hypothesis: RuBisCO likely evolved as a more efficient enzyme when Earth was not yet oxygenated (until ca. 2.6 billion years ago). With rising O2 concentrations through photosynthesis, the enzyme had to become more specific to select against this molecule at the cost of efficiency. Further evolutive improvement of the enzyme seems to be challenging. Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”) Curr. Opin. Biotechnol. 2018, 49:100  Fundamentals in Biology 1, J. Piel The carboxysome, a CO2-enriching bacterial microcompartment Protein container with tightly packed crystalline array of RuBisCO. Likely evolved as response to rising O2 concentration in the atmosphere. ca. 250 RuBisCO molecules Proc. Natl. Acad. Sci. U. S. A. (2012) 109:478 wikimedia commons Carbonic anhydrase Carbonic anhydrase increases the CO2 concentration: Slow equilibrium in water, at CO2 + H2O H+ + HCO3 - 2H+ + CO3 2- physiological pH almost Hydrogen Carbonate exclusively on the side of HCO3- carbonate Reaction catalyzed by carbonic (not a substrate of RuBisCo) anhydrase, (edit in slide master 107-fold acceleration Placeholder for organisational unit name / logo via “View” > “Slide Master”) | |  Fundamentals in Biology 1, J. Piel The CO2 Fixation Step 3-Phospho- glycerate Incorporation of CO2 into the organic molecule 6 CO2 ʘ 12 ATP ribulose 1,5-bisphosphate by the enzyme ribulose 12 1,5-bisphosphate carboxylase (RuBisCO) 12 ADP 6 RuBisCO P ʘ 12 CO2 + Ribulose 1,5- P 6 ADP bisphosphate 12 NADPH Ribulose 1,5- Unstable intermediate 6 ATP bisphosphate 12 NADP+ H2O 12 Pi 6 Glyceraldehyde 12 3-phosphate GAP (GAP) Product: P P 3-Phosphoglycerate 10 (2 equivalents) 4 Pi P Sugars and other biomolecules P 2 GAP Placeholder for organisational unit name / logo (edit in slide master via “View” > “Slide Master”) P | |  Fundamentals in Biology 1, J. Piel Reduction generates the sugar GAP 3-Phospho- glycerate 1. ATP-dependent activation of 3-phosphoglycerate by 6 CO2 phosphorylation, ʘ 12 ATP 12 2. NADPH-dependent reduction to the three-carbon 12 ADP sugar GAP 6 RuBisCO ʘ ATP NADPH 12 -ADP NADP+ Ribulose 1,5- 1,3-Bisphospho- 6 ADP bisphosphate glycerate 3-Phospho- 1,3-Bisphospho- GAP 12 NADPH glycerate glycerate 6 ATP 12 NADP+ See J. Vorholt’s part on substrate-level phosphorylation. 12 Pi This is the reverse sequence of the GAP-DH module: 6 Glyceraldehyde Stryer Biochemistry 9e, Macmillan, Chapter 16 Ø 12 3-phosphate GAP P (GAP) 10 4 Pi Sugars and other biomolecules 1 P 2 GAP Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Closing the circle 3-Phospho- Complex carbon transfer reactions create a C5- from a C3-sugar glycerate Intermediates: C3, C4, C5, C6, and C7 sugars 6 CO2 12 ATP ʘ The same reactions also occur in the pentose phosphate pathway 12 (more later this week) 12 ADP C5 6 RuBisCO GAP C7 Ø C3 P C3 P 12 C6 Ribulose 1,5- 1,3-Bisphospho- P P C4 6 ADP bisphosphate glycerate 12 NADPH 6 ATP 12 NADP+ P P P Fructose-6- Erythrose 4- Sedoheptulose- 12 Pi phosphate phosphate 1,7-bisphosphate 6 Sugar chain Glyceraldehyde C5 Ribose 5- rearrange- GAP 12 3-phosphate phosphate ments GAP C7 C3 (GAP) Ribulose 5- C5 P phosphate C5 10 P P 4 Pi Sugars and other biomolecules 1 P 2 GAP P P P Placeholder for organisational unit name / logo Sedoheptulose (edit in slide master via “View” > “Slide Master”) Ribulose 5- Ribulose 1,5- | | -7-phosphate phosphate bisphosphate  Fundamentals in Biology 1, J. Piel Closing the circle 3-Phospho- glycerate Ø 6 CO2 12 ATP ʘ Transketolase + + 12 12 ADP Fructose GAP Erythrose Xylulose 6-phosphate 4-phosphate 5-phosphate 6 RuBisCO (F6P) (E4P) (Xu5P) 12 Ribulose 1,5- 1,3-Bisphospho- + Aldolase H2 O Pi 6 ADP bisphosphate glycerate Phosphatase 12 NADPH Erythrose Dihydroxy- 4-phosphate acetone (E4P) phosphate Sedoheptulose 1,7-bisphosphate Sedoheptulose 7-phosphate 6 ATP 12 NADP+ (DHAP) (S7P) 12 Pi 6 Sugar chain Glyceraldehyde rearrange- Transketolase + 12 3-phosphate + ments GAP (GAP) Ribulose 5- Sedoheptulose GAP Ribose 5- Xylulose 7-phosphate phosphate 5-phosphate phosphate (S7P) (R5P) (Xu5P) 10 4 Pi Sugars and other Ribulose 5- biomolecules 1 P 2 GAP Placeholder for organisational unit name / logo phosphate | | (edit in slide master via “View” > “Slide Master”) (Ru5P)  Fundamentals in Biology 1, J. Piel Gluconeogenesis converts GAP to Glucose-6-phosphate More details on gluconeogenesis this Thursday 2 GAP Glucose 6-phosphate Gluconeogenesis is mostly a reversal of the glycolysis pathway (see part J. Vorholt). A formal reversal of the Fructose 1,6- Stryer Biochemistry 9e, Macmillan, Fig. 16.2 upper glycolysis pathway generates glucose 6- bisphosphatase phosphate from GAP. Since conversion of ATP to ADP is irreversible, fructose 1,6-phosphatase replaces phosphofructokinase in gluconeogenesis. ʘ Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Day-night cycle in a cyanobacterium Nighttime: Generation of ATP Daytime: Photosynthesis, carbon fixation, and reduced coenzymes biosynthesis (anabolism) (catabolism) CO2 Sugars Sugars Gluconeo- CO2 genesis Nucleotides fixation GAP GAP Glycolysis Amino ATP, Glycolysis acids NADH NADPH ATP H+ ADP NADP+ Fatty Fatty H+ Acetyl-CoA Acetyl-CoA acids acids ATP H2O O2 H+ H+ TCA TCA NAD(P)H, H+ cycle cycle FADH2 Photosynthesis Oxidative phosphoryl- Placeholder for organisational unit name / logo ATP ation | | (edit in slide master via “View” > “Slide Master”) CO2 ATP  Fundamentals in Biology 1, J. Piel Diverse solutions to CO2 fixation have evolved A, Calvin-Benson-Bassham Ø cycle (cyanobacteria, algae, A B C Sci. China Life Sci. 2016, 59: 1106 plants, etc.) B, 3-Hydroxypropionate cycle (green non-sulfur bacteria) G C, Wood-Ljungdahl pathway (anaerobic bacteria and archaea) D, Reductive TCA cycle (green sulfur bacteria) D E F E, Dicarboxylate/4- hydroxybutyrate cycle Two papers report (Archaea) the creation of an artificial pathway by F, 3-Hydroxypropionate/4- synthetic biology hydroxybutyrate cycle and its discovery in (Archaea) nature G, Reductive glycine pathway Currently, seven CO2 fixation pathways are known (Desulfovibrio) Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”) Nat. Commun. 2020, 11:5090 Nat. Chem. Biol. 2020, 16: 538  Fundamentals in Biology 1, J. Piel Examples of symbioses based on carbon fixation “Chlorochromatium aggregatum”: Lichens: Ancient symbiosis: Cyanobacteria-derived a two-bacterial mutualism fungus + photosynthetic chloroplasts of plants and algae bacterium or alga Outer partner: photolithotroph Central partner: movement pxhere.com One of many solar- powered marine sea slugs Corals harboring utilizing chloroplasts of dinoflagellates harboring food algae chloroplasts Brock Chap. 25.1, Fig. 25.1,2 Brock Chap. 25.2, Fig. 25.3,5 Wikimedia commons pixy.org Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Nitrogen fixation: Reduction of atmospheric N2 to ammonia Fixation of atmospheric N2 provides the ammonia needed for biosynthesis of nitrogen-containing metabolites. Occurs in some bacteria and archaea; higher organisms cannot fix nitrogen on their own (but with symbionts). CO2 Sugars Gluconeo- CO2 genesis Nucleotides fixation GAP Amino Glycolysis NH4+ N2 acids NADPH ATP H+ ADP NADP+ Fatty H+ Acetyl-CoA acids Nitrogen fixation module H2O O2 H+ H+ TCA H+ cycle Photosynthesis Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Nitrogen-containing metabolites Amino acids and proteins Nucleotides, nucleic acids Secondary/specialized metabolites Ø Coenzymes Aminosugars (e.g., cell wall biosynthesis) Ø Ø Antibiotic erythromycin Thiamine pyrophosphate Glucosamine Sources of soluble nitrogen are scarce in nature Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel The nitrogen cycle converts forms of inorganic nitrogen The nitrogen cycle: Nitrogen fixation Denitri- fication Nitri- fication Denitrification by soil microbes decreases the concentration of soluble nitrogen – soil loses nitrogen over time. Nitrogen fixation enables life on earth. It is the main biological process that maintains the nitrogen balance in soil for biomass generation in plants and microorganisms. Ammonia incorporation into amino acids is the predominant process by which nitrogen is assimilated (more details Placeholder this week). for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Nitrogen fixation is challenging: human and biological solutions N2 is an extremely stable, apolar, and unreactive molecule (bond energy 940 kJ mol-1). The net reaction N2 → NH4+ is exergonic, but requires a high activation energy to break the bond. The Haber-Bosch process for synthetic nitrogen fixation into ammonia requires high temperature and pressure: 400-450 °C Ø 200 atm iron catalyst Nitrogen-fixing bacteria accomplish the biological reduction at room temperature and atmospheric pressure using the enzyme nitrogenase. Overall reaction catalyzed by nitrogenase: Placeholder for organisational unit name / logo (edit in slide master via “View” > “Slide Master”) ΔG’° = -290.7 kJ/mol Ø | |  Fundamentals in Biology 1, J. Piel Nitrogenase, a remarkable enzyme Electrons for nitrogen reduction are provided by the accessory protein nitrogenase reductase (yellow protein left), which contains an Fe4S4 cluster. The enzyme hydrolyzes ATP to accomplish conformational protein changes and activation during electron transfer to nitrogenase Stryer,Biochemistry, 8th ed. Reductase (Fe protein) Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Nitrogenase, a remarkable enzyme M cluster ʘ Ø Stryer Biochemistry 9e, Macmillan, Fig. 24.3 Reductase Nitrogenase (Fe protein) (MoFe protein) Carbon atom Homocitrate Nitrogenase (MoFe protein left and above) contains two different metal-sulfur clusters (look for the Fe4S4 subclusters for orientation). Alternative enzymes with vanadium or iron centers are known. The FeMo cofactor is unique among all known enzymes Chem. Rev. 2014, 114: 4041 and contains besides iron and molybdenum a single iron- bound carbon atom – a carbide (a formal C4-)! Placeholder for organisational unit name / logo Data support N2 and hydrogen bound to metal atoms, but | | (edit in slide master via “View” > “Slide Master”) the exact mechanism remains unknown.  Fundamentals in Biology 1, J. Piel Examples of nitrogen-fixing organisms (diazotrophs) Termites and cockroaches with Cyanobacteria Azotobacter nitrogen-fixing gut symbionts FEMS Microbiol. Rev-2004, 28:469 wikimedia commons Brock Chap. 18.7, Fig. 18.26 wikimedia commons Trichodesmium spp. cyano- bacteria fix almost 50% of the global ocean nitrogen and give the Red Sea its name. Azotobacter is used as a biological soil fertilizer. Filamentous Anabaena species possess specialized nitrogen-fixing cells termed heterocysts. Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Rhizobia Taxonomically diverse nitrogen-fixing bacteria that colonize root cells of legumes. This triggers the formation of specialized structures termed root nodules. Bacteroids Soy bean root nodules A root nodule contains deformed bacteria induced by rhizobia. called bacteroids. These colonize intracellular plant cell compartments called symbiosomes. Nitrogenase is oxygen-sensitive, but oxygen is needed for ATP generation. The plant produces an oxygen-binding hemoglobin- like protein (leghemoglobin) to decrease the concentration of free (editoxygen in“View” nodules. Placeholder for organisational unit name / logo | | in slide master via > “Slide Master”) Brock Chap. 25.3, Fig. 25. 7,11,14  Fundamentals in Biology 1, J. Piel Summary C and N acquisition Acquired skills: Relationship between catabolism and anabolism Carbon fixation Carbon cycle, main groups of CO2-fixing organisms CBB cycle: input, output, key intermediates RuBisCo and the challenge of CO2 fixation Gluconeogenesis Nitrogen fixation Nitrogen cycle, main groups of N2-fixing organisms Nitrogenase and the challenge of N2 fixation Important organisms, symbiosis based on diazotrophy Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel Extra Slide (not Exam-Relevant) for Explanation Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)  Fundamentals in Biology 1, J. Piel The carboxysome, a CO2-enriching bacterial microcompartment Ø How does carbonic anhydrase increase the CO2 concentration? 1. A slow equilibrium exists in water between free CO2 and 3. Carbonic anhydrase accelerates the CO2/HCO3- forms of carbonate: equilibrium 107-fold to generate free CO2 CO2 + H2O H+ + HCO3- 2H+ + CO32- CO2 + H2O H+ + HCO3- Hydrogen Carbonate carbonate Reaction catalyzed by 2. This limits the availability of free CO2, the only form that carbonic anhydrase RuBisCO can use: Proc. Natl. Acad. Sci. U. S. A. (2012) 109:478 RuBisCO can access CO2 CO2 HCO3- CO32- the carbon Front. Plant Sci. (2013) 4: 140 Access blocked HCO3- Placeholder for organisational unit name / logo | | (edit in slide master via “View” > “Slide Master”)