Nitrogen Acquisition and Amino Acid Metabolism

Chapter 26 Nitrogen Acquisition and Amino Acid Metabolism Outline • 26.1 The Two Major Pathways of N Acquisition • 26.2 The Fate of Ammonium • 26.3 Glutamine Synthetase • 26.4 Amino Acid Biosynthesis • 26.5 Metabolic Degradation of Amino Acids Major Pathways for N Acquisition • All biological compounds contain N in a reduced form • The principal inorganic forms of N are in an oxidized state • Thus, N acquisition must involve reduction of the oxidized forms (N2 and NO3-) to NH4+ • Nearly all of this is in microorganisms and green plants Animals gain N through diet Figure 26.1- The nitrogen cycle Organic N compounds are formed by the incorporation of NH4+ into C skeletons • Ammonium can be formed from oxidized inorganic precursors by reductive reactions: – nitrogen fixation reduces N2 to NH4+; – nitrate assimilation reduces NO3- to NH4+ • Nitrifying bacteria can oxidize NH4+ back to NO3- and obtain energy for growth in the process of nitrification • Denitrification is a form of bacterial respiration whereby nitrogen oxides serve as electron acceptors in the place of O2 under anaerobic conditions Overview of N Acquisition Nitrogen assimilation and nitrogen fixation • Nitrate assimilation occurs in two steps: – 2e- reduction of nitrate to nitrite and – 6e- reduction of nitrite to ammonium • Nitrate assimilation accounts for 99% of N acquisition by the biosphere • Nitrogen fixation involves reduction of N2 in prokaryotes by nitrogenase Nitrate Assimilation Electrons are transferred from NADH to nitrate • Pathway involves -SH of enzyme, FAD, cytochrome b and MoCo - all protein-bound • Nitrate reductases are big - 210-270 kD • See Figure 26.2 for MoCo structure • MoCo required both for: – reductase activity – assembly of enzyme subunits to active dimer • • Figure 26.2 The novel prosthetic groups of nitrate reductase and nitrite reductase (a) The molybdenum cofactor of nitrate reductase The molybdenum-free version of this compound is a pterin derivative called molybdopterin (b) Siroheme, a uroporphyrin derivative, is a member of the isobacteriochlorin class of hemes, a group of porphyrins in which adjacent pyrrole rings are reduced Siroheme is novel in having carboxylate-containing side chains These carboxylate groups may act as H+ donors during the reduction of NO2- to H4+ Nitrite Reductase Light drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and then to nitrite • See Figure 26.2b for siroheme structure • Nitrite is reduced to ammonium while still bound to siroheme • In higher plants: – nitrite reductase is in chloroplasts, – nitrate reductase is cytosolic Figure 26.3- Domain organization within the enzymes of nitrate assimilation • The numbers denote residue number along the a.a sequence of the proteins • The numbering for nitrate reductase is that from the green plant Arabidopsis thaliana; • the plant nitrite reductase sequence shown here is spinach; • the fungal nitrite reductase is Neurospora crassa Enzymology of N fixation Only occurs in certain prokaryotes • Rhizobia fix nitrogen in symbiotic association with leguminous plants: – Rhizobia fix N for the plant – plant provides Rhizobia with carbon substrates • All nitrogen fixing systems appear to be identical, requiring: – – – – – nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions, regulatory controls (ADP inhibits and NH4+ inhibits expression of nif genes Degradation of Amino Acids The 20 aa are degraded to produce (mostly) TCA intermediates • Know the classifications of aa (Fig 26.41) • Know which are glucogenic and ketogenic • Know which are purely ketogenic Figure 26.41 · Metabolic degradation of the common aa The 20 common aas can be classified according to their degradation products Those that give rise to precursors for glucose synthesis, such as aketoglutarate, succinylCoA, fumarate, oxaloacetate, and pyruvate, are termed glucogenic (shown in pink) Those degraded to acetyl-CoA or acetoacetate are called ketogenic (shown in blue) because they can be converted to fatty acids or ketone bodies Some aas are both glucogenic and ketogenic • Figure 26.42 · Formation of pyruvate from alanine, serine, cysteine, glycine, tryptophan, or threonine Nitrogen Excretion • Animals often enjoy a dietary surplus of N • Excess N liberated upon metabolic degradation of aas is excreted by animals in different ways, in accord with the availability of water Aquatic animals simply release free ammonia to the surrounding water; such animals are termed ammonotelic (from the Greek telos, end) Terrestrial and aerial species employ mechanisms that convert ammonium to less toxic waste compounds that require little H2O for excretion Many terrestrial vertebrates are ureotelic (they excrete excess N as urea (formed via the urea cycle)- a highly watersoluble nonionic substance) The uricotelic organisms conver N to uric acid, a rather insoluble purine analog Birds and reptiles are uricoteles (Uric acid metabolism is discussed in the next chapter) • Some animals can switch from ammonotelic to ureotelic or to uricotelic metabolism, depending on water availability ... between nitrogenase reductase and a nitrogenase ab dimer Within the nitrogenase reductase (shown in green and yellow here), the Fe4S4 center is closest to the nitrogenase ab dimer The nitrogenase... kinase Amino Acid Biosynthesis • Plants and microorganisms can make all 20 amino acids and all other needed N metabolites • In these organisms, Glu is the source of N, via transamination (aminotransferase)... Pathways of N Acquisition • 26.2 The Fate of Ammonium • 26.3 Glutamine Synthetase • 26.4 Amino Acid Biosynthesis • 26.5 Metabolic Degradation of Amino Acids Major Pathways for N Acquisition •