February 12, 2002 Chapter 26 Nitrogen Acquisition Biochemistry 432/832 February 12, 2002 Chapter 26 Nitrogen Acquisition

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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.

The nitrogen cycle

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

Electrons are transferred from NADH to nitrate 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 kDa MoCo required both for reductase activity and for assembly of enzyme subunits to active dimer

Novel prosthetic groups used in N acquisition Molybdopterin Siroheme

Mo-containing enzymes Mo is the heaviest element used by eukaryotes Two classes of molybdoenzymes 1) Molybdopterin-dependent enzymes Nitrate reductase Formate dehydrogenase Aldehyde oxidase Xanthine dehydrogenase Sulfate oxidase 2) Nitrogenase Molybdopterin

Nitrite Reductase Light drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and then to nitrite Nitrite is reduced to ammonium while still bound to siroheme In higher plants, nitrite reductase is in chloroplasts, but nitrate reductase is cytosolic

Enzymology of N fixation Only occurs in certain prokaryotes Rhizobia fix nitrogen in symbiotic association with plants Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates All nitrogen fixing systems are very similar They require nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions and regulatory controls (ADP inhibits reaction and NH4+ inhibit expression of nif genes)

Two protein components: nitrogenase reductase and nitrogenase Nitrogenase Complex Two protein components: nitrogenase reductase and nitrogenase Nitrogenase reductase is a 60 kDa homodimer with a single 4Fe-4S cluster Very oxygen-sensitive Binds MgATP 4ATP required per pair of electrons transferred Reduction of N2 to 2NH3 + H2 requires 4 pairs of electrons, so 16 ATP are consumed per N2

Why should nitrogenase need ATP??? N2 reduction to ammonia is thermodynamically favorable However, the activation barrier for breaking the N-N triple bond is enormous 16 ATP provide the needed activation energy

To break the triple bond, energy input in necessary

Nitrogenase A 220 kDa heterotetramer Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters, etc.) Four 4Fe-4S clusters plus two FeMoCo, an iron-molybdenum cofactor Nitrogenase is slow - 12 e- pairs per second, i.e., only three molecules of N2 per second

Structures of two types of metal clusters in nitrogenase: The P-cluster FeMoCo

The nitrogenase reaction Accumulation of electrons

Complex between nitrogenase reductase and nitrogenase

Regulation of nitrogen fixation ADP inhibits NH4+ represses expression ADP-ribosylation inhibits

Three major reactions in all cells The Fate of Ammonium Three major reactions in all cells Carbamoyl-phosphate synthetase two ATP required - one to activate bicarbonate, one to phosphorylate carbamate Glutamate dehydrogenase reductive amination of alpha-ketoglutarate to form glutamate Glutamine synthetase ATP-dependent amidation of gamma-carboxyl of glutamate to glutamine

The glutamate dehydrogenase reaction

The glutamine synthetase reaction

Ammonium Assimilation Two principal pathways Principal route: GDH/GS in organisms rich in N both steps assimilate N Secondary route: GS/GOGAT in organisms confronting N limitation GOGAT is glutamate synthase or glutamate:oxo-glutarate amino transferase

The glutamate dehydrogenase/glutamine synthase pathway Two N fixing steps - one inefficient One each

The glutamate synthase reaction

The glutamine synthase/GOGAT pathway One N fixing step - inefficient but expensive One NADPH Two ATP

Glutamine Synthetase A Case Study in Regulation GS in E. coli is regulated in three ways: Feedback inhibition Covalent modification (interconverts between inactive and active forms) Regulation of gene expression and protein synthesis - - control the amount of GS in cells

The glutamine synthetase reaction

Glutamine synthetase structure stack of two hexagons

Allosteric Regulation of Glutamine Synthetase Nine different feedback inhibitors: Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P and glucosamine-6-P Gly, Ala, Ser are indicators of amino acid metabolism in cells Other six are end products of biochemical pathways This effectively controls glutamine’s contributions to metabolism

Allosteric regulation of glutamine synthase activity by feedback inhibition

Covalent Modification of Glutamine Synthetase Each subunit is adenylylated at Tyr-397 Adenylylation inactivates GS Adenylyl transferase catalyzes both the adenylylation and deadenylylation PII (regulatory protein) controls both activities AT:PIIA catalyzes adenylylation AT:PIID catalyzes deadenylylation -ketoglutarate and Gln also affect

Covalent modification of glutamine synthase - adenylylation of Tyr397