Chapter 22 Biosynthesis of amino acids, nucleotides and related molecules 1. Reduction (fixation) of N 2 into ammonia (NH 3 or NH 4 + ) 2. Synthesis of the 20 amino acids. 3. Synthesis of other biomolecules from amino acids 4. The de novo pathways for purine and pyrimidine biosynthesis. 5. The salvage pathways for purine and pyrimidine reuse.

1. The nitrogenase complex in certain bacteria (diazotrophs, 固氮生物 ) catalyzes the conversion of N 2 to NH 3 The nitrogen in amino acids, purines, pyrimidines and other biomolecules ultimately comes from atmospheric nitrogen. Cyanobacteria ( 蓝藻细菌, photosynthetic) and rhizobia ( 根 瘤菌, symbiont) can fix N 2 into NH 3. The reduction of N 2 to NH 3 is thermodynamically favorable : N 2 + 6e - + 6H + 2NH 3  G` o = -33.5kJ/mol But kinetically unfavorable: the bond energy for the triple bond in N 2 is 942 kJ/mol.

The nitrogenase ( 固氮酶 ) complex mainly consists of two types of enzymes: the dinitrogenase and the dinitrogenase reductase. The dinitrogenase (containing molybdenum ，钼, thus called the MoFe protein) is a tetramer of two different subunits, containing multiple 4Fe-4S centers and two Mo-Fe clusters. The dinitrogenase reductase (also called the Fe protein) is a dimer of two identifcal subunits, containing a single 4Fe-4S redox center. The nitrogenase complex is highly conserved among different diazotrophs.

Cyanobacteria and Rhizobia can fix N 2 into ammonia Rhizobia exist in nodules of leguminous plants

The nitrogenase complex The dinitrogenase (tetramer) The dinitrogenase reductase (dimer) The dinitrogenase reductase (dimer) ADP 4Fe-4S (P-cluster) Fe-Mo cofactor 3Fe-3S Mo

2. Electrons are transferred through a series of carriers to N 2 for its reduction on the nitrogenase complex Eight electrons are believed to be needed for each round of fixation reaction: with six for reducing one N 2 and two for reducing 2 H + (to form H 2 ). The electrons mainly come from reduced ferredoxin (from photophosphorylation) or reduced flavodoxin (from oxidative phosphorylation) and are transferred to dinitrogenase via dinitrogenase reductase.

For each electron to be transferred from dinitrogenase reductase to dinitrogenase, two ATPs are hydrolyzed causing a conformational change which reduces the electron affinity for the reductase (i.e., an increased reducing power). The oxidized and reduced dinitrogenase reductase dissociates from and associates with the dinitrogenase, respectively. The nitrogenase is in imperfect enzyme: H 2 is formed along with NH 3.

The overall reaction catalyzed is: N 2 + 8H + +8e ATP + 16H 2 O  2NH 3 + H ADP + 16P i

Electrons are transferred to N 2 bound in the active site of dinitrogenase via ferredoxin/flavodoxin and dinitrogenase reductase

N 2 is believed to bind to the cavity of the Fe-Mo cofactor of the dinitrogenase active site. 3Fe-3S

3. The nitrogenase complex is extremely labile to O 2 and various protective mechanisms have evolved Some diazotrophs exist only anaerobically. Some cyanobacterial cells develop thick walls to prevent O 2 from entering. The bacteria in root nodules are isolated from O 2 by being bathed in a solution of the oxygen-binding protein leghemoglobin.

Leghemoglobin, produced in legume plants, has a high affinity to O 2 and protects the nitrogenase complex in rhizobia

4. Reduced nitrogen in the form of NH 4 + is assimilated into amino acids via a two-enzyme pathway First NH 4 + is added to the side chain of glutamate to form glutamine in an ATP-dependent reaction catalyzed by glutamine synthetase. Then the side chain amino group of Gln is further transferred to  -ketoglutarate to form Glu in a reaction catalyzed by glutamate synthase, an enzyme only present in bacteria and plants, not in animals.

The amide group of Gln is a source of nitrogen in the synthesis of a variety of compounds, such as carbamoyl phosphate, Trp, His, glucosamine-6-P, CTP, and AMP. The amino groups of most other amino acids are derived from glutamate via transamination.

Newly fixed nitrogen in the form of NH 4 + is first incorporated into glutamate to form glutamine

e The side chain amino group of glutamine is then transferred to  -ketoglutarate to form Glu

5. The bacterial Glutamine synthetase is a central control point in nitrogen metabolism The bacterial glutamine synthetase has 12 identical subunits (each having an independent active site) arranged as two hexagonal rings. Each subunit of the enzyme is accumulatively inhibited by at least eight allosteric effectors.

In addition the enzyme is more susceptible to the allosteric inhibition by having Tyr 397 residue modified by adenylylation. The addition and removal of the AMP group to the glutamine synthetase are catalyzed by two active sites of the same bifunctional adenylyltransferase (AT). The substrate specificity of AT was found to be controlled by a regulatory protein, PII.

The activity of PII, in turn, is regulated by the uridylylation of a specific Tyr residue: PII-UMP stimulates the adenylylation activity of AT, however, the unmodified PII stimulates the deadenylylation activity of AT. The addition and removal of UMP to PII, in turn, are again catalyzed by two active sites of the same protein, uridylyltransferase (UT):  -ketoglutarate and ATP stimulate the uridylylation, however, Gln and P i stimulate the deuridylylation (thus adenylylation of AT, inactivating glutamine synthetase).

The bacterial glutamine synthetase has 12 subunits arranged as two rings of hexamers Active sites Tyr 397 (adenylylation site)

The glutamine synthetase is accumulatively inhibited by at least 8 allosteric effectors, mostly end products of glutamine metabolism

A specific Tyr residue in bacterial glutamine synthetase can be reversibly adenylylated

6. Amidotransferases catalyze the transfer of the amide amino group from Gln to other compounds All of the enzymes contain two structural domains: one binding Gln, the other binding the amino group accepting substrate. A Cys residue is believed to act as a nucleophile to cleave the amide bond, forming a covalent glutamyl- enzyme intermediate with the NH 3 produced remain in the active site and react with the second substrate to form an aminated product.

A proposed action mechanism for amidotransferases

7. The 20 amino acids are synthesized from intermediates of glycolysis, the citric acid cycle, or pentose phosphate pathway The nitrogen is provided by Glu and Gln. As in amino acid degradation, some of the synthetic pathways (e.g., for Ala, Glu, and Asp) are very simple, but others (e.g., the aromatic ones) are very complex. Most bacteria and plants can synthesize all 20, but mammals can only synthesize about 10, with 10 being “essential”, needed in the diet.

The pathways for the biosynthesis of the 20 amino acids can be categorized into six families according to the metabolic precursors used.

Intermediates of glycolysis, the citric acid cycle, and pentose phosphate pathway serve as precursors for synthesizing the 20 standard amino acids. + acetyl-CoA + pyruvate +Asp + ATP Gln

8.  -ketoglutarate is the common precursor for the biosynthesis of Glu, Gln, Pro, and Arg in bacteria To make Glu, the amino group can be from the side chain of Gln (catalyzed by an amidotransferase, glutamate synthase in bacteria) or ammonia (catalyzed by glutamate dehydrogenase in vertebrates). Gln is made from Glu by obtaining an amino group from ammonia in a reaction catalyzed by glutamine synthetase.

Proline is synthesized from Glu via one step of phosphorylation (activation), two steps of enzyme- catalyzed reductions (using NADPH) and one step of nonenzymatic cyclization reaction (forming a Schiff base). To make Arg, ornithine is first made from Glu with a few steps similar to that of Pro synthesis, except that an acetyl group is first added to the  -amino group of glutamate to protect the spontaneous Schiff base formation between the aldehyde group and the amino group and removed after an amino group is transferred to the aldehyde group.

Arg is then synthesized from ornithine using steps similar to those of urea cycle in bacteria. Arg from the diet can be converted to Pro in mammals by being converted to ornithine first using the urea cycle enzymes and then to  1 -pyrroline-5- carboxylate by ornithine  -aminotransferase. Similarly, Arg can be formed from glutamate  - semialdehyde (an intermediate of Pro synthesis) also by using ornithine  -aminotransferase.

Glu is synthesized from  -ketoglutarate by an amination reaction with amino groups obtained from Gln or ammonia

Gln is synthesized from Glu by obtaining an amino group from ammonia in a reaction catalyzed by glutamine synthetase Glutamine synthetase

Glutamamate kinase Glutamate dehydrogenase spontaneous Pyrroline carboxylate reductase aminotransferase Acetylglutamate synthase

9. Ser, Gly, and Cys are all derived from 3-phosphoglycerate Ser is synthesized (in all organisms) from 3- phosphoglycerate via NAD + -dependent oxidation, PLP-dependent transamination, and dephosphorylation reactions. Gly is either derived from Ser by removal of the side chain  -carbon (catalyzed by serine hydroxymethyltransferase, using PLP and H 4 folate) or synthesized (in vertebrate liver cells) from CO 2 and NH 4 + (catalyzed by glycine synthase, using N 5,N 10 -methylene H 4 folate).

In plants and bacteria, Cys is made from Ser by replacing the side chain –OH group (after being activated first by acetylation) by an –SH group using sulfide ( 硫化物 ) reduced from sulfate ( 硫酸盐 ). In mammals, Cys is made from Met (providing the sulfur atom after being converted to homocysteine) and Ser (providing the carbon skeleton).

Ser is synthesized from 3-phosphoglycerate via oxidation, transamination, and dephosphorylation reactions Phosphoserine aminotransferase

Gly is either derived from Ser by removal of the side chain  -carbon or synthesized (in vertebrate liver cells) from CO 2 and NH 4 + Serine hydroxymethyl transferase Glycine Synthase

Cys is derived from Ser via a two-step pathway using sulfide in plants and bacteria

Cys is synthesized from homocysteine (which is derived from Met) and Ser in mammals ( 胱硫醚  - 合成酶 ) ( 胱硫醚  - 裂解酶 ) Homocysteine ( 高半胱氨酸）

Homocysteine is derived from Met via S-adenosylmethionine and S- adenosylhomocysteine intermediates Methionine adenosyl transferase Methyl transferase Methionine synthase Hydrolase Homocysteine MetATP

10. Ala, Val, and Leu are derived from pyruvate Ala is synthesized from pyruvate via a simple transamination reaction (with amino group donated by Glu). Val is synthesized from the transferring of a two- carbon unit to a pyruvate, forming  -acetolactate, which is then converted via isomerization, reduction, dehydration, and transamination.

The immediate precursor of Val,  -keto- isovalerate, is converted to Leu via four steps of reactions: the addition of an acetyl group, a position switching of an –OH group, an oxidative decarboxylation, and a transamination reaction.

Acetohydroxy acid synthase Acetohydroxyl acid isomeroreductase Dihydroxy acid dehydratase Valine aminotransferase Val is synthesized from two pyruvates

Leu is synthesized from  -keto-isovalerate Synthase Isomerase Dehydrogenase Aminotransferase Leu

11. Asp, Asn, Thr, Met are all derived from oxaloacetate Asp is synthesized from oxaloacetate via a simple transamination reaction. Asn is synthesized from Asp by an amidation ( 酰胺 化 ) reaction with Gln donating the NH 4 + (catalyzed by an amidotransferase). Thr and Met are all derived from Asp, branching off after Asp is converted to aspartate  -semialdehyde (in a way similar to Glu to glutamate  - semialdehyde conversion).

Thr is synthesized from aspartate  - semialdehyde via homoserine. Met is also synthesized from homoserine: it is first activated via succinylation; the accepting the sulfur atom from cysteine and methyl group from N 5 -methyl H 4 folate.

Asp is synthesized from oxalocaetate by a simple transamination Transaminase

Asn is synthesized from Asp by transferring an amino group from the amide group of Gln Asparagine synthetase

Asp is first converted to aspartate-  -semialdehyde which is then used to synthesize Thr, Met, and Lys. Homoserine dehydrogenase Homoserine kinase Threonine synthase Thr is synthesized from Asp via homoserine

Homoserine acyltransferase Cystathionine-  -synthase Cystathionine-  -lyase Methionine syntase Met is also synthesized from homoserine via cystathionine and homocysteine Met

12. Lys and Ile are derived from oxaloacetate and pyruvate Lys is synthesized from aspartate  -semialdehyde (which is derived from Asp) and pyruvate (contributing to the two carbons at positions of  and  of Lys), with glutamate donating the  -amino group. Ile is made from  -ketobutyrate (the dehydration/deamination product of Thr) and pyruvate, using the same set of enzymes as that for Val synthesis.

Dihydropicolinate synthase Dihydropicolinate synthase Lys is synthesized from aspartate-  -semialdehyde and pyruvate Dehydrogenase

Synthase Aminotransferase Obtaining the  -amino group from Glu

Desuccinylase Epimerase Decarboxylase Lys Removing the carboxyl group of the condensed pyruvate

Acetolactate synthase Acetolactate syntase Acetohydroxy acid isomeroreductase Acetohydroxy acid isomeroreductase Dihydroxyl acid dehydratase Valine aminotransferase Pyruvate Val  isopropylmalate synthase Isopropylmalate isomerase Dehydrogenase Leucine aminotransferase Leu Ile Thr Ile is made from Thr and Pyruvate using the same set Of enzymes for Val synthesis

13. The aromatic amino acids are derived from phophoenoylpyruvate and erythrose 4-phosphate All aromatic amino acids share the first seven reactions for biosynthesis, up to the formation of chorismate ( 分支酸 ). All the carbons of the aromatic ring are derived from PEP and erythrose 4-P: ring closure follows the condensation reaction; which in turn is followed by step-wise double bond introduction (by dehydration).

A second PEP enters to make chorismate. Chorismate can be converted to anthranilate (by accepting an amino group from Gln and releasing a pyruvate) or prephenate (by switching the position of the PEP component via a Claisen rearrangement) by the catalysis of anthranilate synthase or chorismate mutase respectively. Anthranilate ( 邻氨基苯甲酸 ) can be further converted to Trp via another four steps of reaction: accepting two carbons from 5-phosphoribosyl-1- pyrophosphate (PRPP), and three carbons from Ser.

Prephenate ( 预苯酸 ) can be converted to Phe by releasing the carboxyl and hydroxyl groups from the ring, and accepting an amino group from Glu via a transamination reaction. Prephenate can also be converted to Tyr by an oxidative decarboxylation and a transamination reaction.

four steps three steps one step four steps two steps The biosynthesis of all aromatic amino acids share the first seven steps of reactions

The 2 nd PEP enter here Ring formation step 1st double bond 2 nd double bond Chorismate Chorismate, formed from the condensed product of PEP and erythrose 4-P, is the common precursor of Trp, Tyr and Phe.

Chorismate is converted to Trp by accepting an amino group from Gln, two carbons from 5 phosphoribosyl- 1-pyrophosphate (PRPP), and three carbons from Ser. ( 邻氨基苯甲酸 ) Synthase Transferase Isomerase Synthase Trp Synthase

Chorismate can also be converted to prephenate which serve as the common precursor of Phe and Tyr biosynthesis Claison rearrangement Chorismate mutase Dehydrogenase dehydratase

14. Tryptophan synthase catalyzes the conversion of indole-3-glycerol phosphate to Trp with indole as an intermediate The tetrameric enzyme has two  and two  subunits. Indole-3-glycerol phosphate enters the  subunits (the “lyase”) and is cleaved to form indole and glyceraldehyde-3-P. Serine enters the  subunits, forms a Schiff base with PLP, and is dehydrated to form a PLP- aminoacrylate adduct.

The indole intermediate formed on the a subunit then enters the active site of the  subunit via a “substrate channel”, where it condenses with the PLP-aminoacylate intermediate to form a ketimine ( 酮亚胺 ) intermediate, which is then converted to a aldimine ( 醛亚胺 ) intermediate. The aldimine intermediate is then hydrolyzed to form Trp.

The  and  subunits of tryptophan synthase have different enzymatic activities with substrate channeling A ketimine An aldimine

The hydrophobic indole is channeled from the  to the  subunits in tryptophan synthase

15. Histidine is derived from three precursors: 5-phosphoribosyl 1- pyrophosphate, ATP and Gln The synthesis begins with the condensation of ATP and PRPP: N-1of the purine ring is linked to the C-1 of the ribose. For the final synthesis of Histidine, PRPP contributes five carbons, ATP contributes one nitrogen and one carbon (both from the purine ring), Gln contributes one nitrogen, and Glu contributes the 3 rd nitrogen (of the  -amino group).

The unusual synthesis of histidine from a purine is taken as evidence supporting the hypothesis that life originated from RNA: the synthetic pathway is considered as a “fossil” of the transition from RNA to the more efficient protein-based life forms.

Transferase Hydrolase The synthesis of histidine begins with the condensation of PRPP and ATP: The C-1 of the ribose ring is linked to the N-1 of the purine ring.

Hydrolase Isomerase Aldose Ketose Amidotransferase AminotransferaseDehydrogenase Dehydratase Histidine is made from PRPP, ATP, Gln and Glu. Ring opens between N-1 and C-6 Cleaved between C-2 and N-3 A purine!

16. Several feedback inhibition mechanisms are found to work in regulating amino acid biosynthesis The first case of allosteric feedback inhibition was discovered in studying Ile biosynthesis in E. coli The enzyme catalyzing the first reaction (threonine dehydratase) is inhibited by the end product (Ile) in a synthetic pathway. Several intricate feedback inhibition mechansims have been found in branched pathways for amino acid biosyntheses.

In enzyme multiplicity, several isozymes are present to catalyze a common step of reaction: each isozyme responds to a different allosteric modulator, avoiding the inhibition of a common reaction by only one end product. In concerted inhibition, one enzyme is inhibited by two or more than two modulators, with effect more than additive. In sequential feedback inhibition, the end products inhibit enzymes catalyzing the branching points only, while the initial committing step is inhibited by common precursors of the end products.

The first case of allosteric feedback inhibition was discovered in studying Ile synthesis in E. coli

In branching pathways leading to the synthesis of multiple end products from common precursors, several forms of feedback inhibition mechanisms are used to coordinately regulate the synthesis of all the amino acids.

Several intricate feedback inhibition mechansims have been found in branched pathways for amino acid biosyntheses Sequential Concerted Enzyme multiplicity High level of one (e.g., Y) should not prevent the synthesis of the other (e.g., Z).