Nucleotide biosynthesis

Presentazione sul tema: "Nucleotide biosynthesis"— Transcript della presentazione:

1 Nucleotide biosynthesis
Base Nucleoside Nucleotide (base + ribose) (nucleoside + phosphate) Adenine Adenosine AMP (adenylic acid or adenosine monophosphate) Guanine Guanosine GMP (guanylic acid or guanosine monophosphate) Uracil Uridine UMP (uridylic acid or uridine monophosphate) Thymine Thymidine TMP (thymidylic acid or thymidine monosphosphate) Cytosine Cytidine CMP (cytidylic acid or cytidine monophosphate)

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3 Orotico aciduria Malattia genetica rara caratterizzata da anemia per difetto di maturazione degli eritrociti e per la presenza di acido orotico nelle urine. I soggetti affetti sono incapaci di convertire l’acido orotico in UMP Tipo 1: carenza dell’enzima UMP sintasi (sia orotato fosforibosile transferasi che orotato monofosfato decarbossilasi) Tipo 2: difetto a carico solo dell’ orotato monofosfato decarbossilasi (terapia con uridina) Dosaggio acido orotico nelle urine HPLC 275 nm Più specifico LC/MS/MS

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5 Experiments with radioactive tracers showed that the purine ring is assembled from 5 different types of building block CO2 Glycine Amino group of aspartate N C N10-formyl-THF N10-formyl-THF Amide N of glutamine (THF = tetrahydrofolate) A common pathway of 11 steps beginning with phosphoribosyl-pyrophosphate (PRPP) and glutamine leads to inosine monophosphate (IMP). Separate branches then lead to AMP and GMP. PRPP + Gln AMP IMP GMP

6 1 PRPP sintasi 2 amidofosforibosile transferasi 3 GAR sintetasi 4 GAR transformilasi 5 FGAM sintasi 6 AIR sintasi 7 AIR carbossilasi 8 SACAIR sintetasi 9 Adenilsuccinatoliasi 10 AICAR transformilasi 11 IMP sintasi

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8 Biosintesi dei deossiribonucleotidi e catabolismo delle purine

9 ribonucleotide reductase
ADP, GDP, CDP & UDP can be reduced to the corresponding deoxyribonucleotides ADP GDP CDP UDP dADP dGDP dCDP dUDP H2O X SH ribonucleotide reductase X S dTTP Nucleoside diphosphates are reduced by cysteines in the enzyme, ribonucleotide reductase. NADP+ NADPH DNA replication also requires thymine, which is made from dUDP. Regeneration of the reduced enzyme requires several steps)

10 Primates oxidize purines to uric acid for excretion
NH2 C N CH HC ribose C HN N CH O H2N-C ribose O C HN N C OH HO H uric acid C OH N C OH HO H adenosine guanosine Birds, reptiles and insects also excrete uric acid. Fish and most terrestrial mammals oxidize uric acid further before excreting the product. C HN C O O N H uric acid has several tautomeric forms

11 Uric acid production from purines
adenosine inosine hypoxanthine NH2 C N CH HC ribose ribose C HN N CH O HC C HN N CH O HC H H2O NH4+ Pi ribose-1-P adenosine deaminase C HN N CH O H2N-C ribose C HN N CH O H HO O2 + H2O H2O2 xanthine oxidase guanosine xanthine uric acid O2 + H2O C HN N C OH O HO H xanthine oxidase H2O2 Xanthine oxidase contains FAD, a Mo complex, and two Fe-S centers. Its substrates are the free purines, not nucleosides or nucleotides.

12 Mutations in adenosine deaminase cause Severe Combined Immunodeficiency Disease SCIDS
deoxy-adenosine NH2 C N CH HC deoxyribose H2O NH4+ deoxyribose C HN N CH O HC uric acid adenosine deaminase In the absence of adenosine deaminase, dATP accumulates to high levels. The dATP inhibits ribonucleotide reductase, preventing synthesis of the other deoxynucleotides and cutting off DNA synthesis. Lymphocytes are particularly susceptible to this inhibition. Untreated SCID is fatal. SCID was one of the first disorders to be addressed by gene therapy. dATP - ribonucleotide reductase ribonucleotides deoxyribonucleotides DNA

13 Deposition of sodium urate crystals in tissues causes gout
Impaired excretion of uric acid results in high levels of uric acid in body fluids. Crystals of sodium urate form in the toe, kidney and other tissues, causing painful inflamation. uric acid hypoxanthine xanthine C HN N C OH O HO H CH HC O2 + H2O H2O2 xanthine oxidase C HN N O HC H C HN N C O- O HO H Na+ Gout can be treated with allopurinol, an analog of hypoxanthine that inhibits xanthine oxidase. allopurinol sodium urate

14 • Gotta: dal latino «gutta » (= goccia)
• Già descritta da Ippocrate (ca. 460 – ca. 370 a.C.), Galeno (130 – 201 d.C.) e Paracelso (1493 – 1541) • Definizione: – Artropatia cristallina con deposito intra- ed extrarticolare di cristalli di acido urico, in presenza di un difetto del metabolismo delle purine • Deposito di cristalli di acido urico: – Nelle articolazioni → «vera» artropatia urica – Nei tessuti → possibile formazione di tofi (noduli) • Spesso associata ad una nefropatia urica, disturbi del metabolismo del glucosio e/o dei grassi

15 Diagnostica • Laboratorio
– Puntato: tipici cristalli di acido urico, evidenziati al microscopio con luce polarizzata (rifrazione neg.) – Escissione di tofi delle parti molli: valutazione al microscopio del materiale biancastro estratto e dimostrazione di cristalli di acido urico in massa – Sangue: nell’attacco acuto di gotta nel 90% dei casi vi è un’iperuricemia. Inoltre aumento dei parametri fiammatori (VES e PCR↑, leucocitosi)

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18 Esami necessari per chiarire la causa di un’iperuricemia
– Acido urico sierico – Creatinina sierica – Eliminazione dell’acido urico e della creatinina nelle urine delle 24 h (clearance)

19 “Salvage” pathways capture free purine and pyrimidine bases and regenerate nucleotides
adenine phosphoribosyl transferase NH2 C N CH HC H NH2 C N CH HC OH O P O-CH2 adenine O O- OPOPO- OH P O-CH2 5-phosphoribosyl-1-pyrophosphate (PRPP) PPi AMP Another enzyme (hypoxanthine-guanine phosphoribosyl transferase) works on hypoxanthine and guanine. Mutations in this enzyme lead to Lesch-Nyhan syndrome.

20 Lesch-Nyhan syndrome Lesch-Nhyan syndrome, an X-linked disorder that results from a mutation in the gene for hypoxanthine-guanine ribosylphosphotransferase, is characterized by severe neurological defects. mental retardation self-mutilation cerebral palsy elevated uric acid (gout)

21 Lesch-Nyhan Syndrome can be diagnosed prenatally
Fetal fibroblasts obtained by amniocentesis are cultured in the presence of 3H-hypoxanthine. Cells from normal individuals convert hypoxanthine into IMP, which procedes to AMP and GMP and is incorporated into DNA. normal C HN N CH O HC H OH O P O-CH2 C HN N CH HC PRPP PPi hypoxanthine Lesch-Nyhan IMP Blocked in Lesch-Nyhan syndrome AMP GMP DNA