1. ALCOHOL DEHYDROGENASE (ADH) Iñaki Mtz. de Ilarduya Lorena Pantano Rubiño Albert Mascarell i Creus Structural Bioinformatics (ADH Speach) Msc. Bioinformatics -5 February 2007-

2. INDEX ● 1. INTRODCUTION – 1.1 Structural Classification (SCOP) ● N-ter (GroES-like fold) ● C-ter ( NAD(P)-binding Rossmann-fold) – 1.2 Functions – 1.3 Metabolic Pathways involved – 1.4 ADH System overview ● 2. STRUCTURAL FEATURES ● 3. CONSERVATION AND EVOLUTION

3. 1.1 Structural Classification (SCOP) Root: scop Class: All beta proteins Fold: GroES-like Contains barrel, partly opened; n*=4, S*=8; meander Superfamily: GroES-like Family: Alcohol dehydrogenase-like, (N-terminal domain) Protein: Alcohol dehydrogenase (contains a Zn-finger subdomain, residues 94-117) Species: Human (Homo sapiens), different isozymes

4. 1.1 Structural Classification N-ter domain GroES-like ● GroES-like fold: – Irregular Beta Barrel – Four β-strands n=4 – Shear number = 8 – Insertion of a short 3 10 helix before the third strand of the β-barrel

5. 1.1 Structural Classification (SCOP) Root: scop Class: Alpha and beta proteins (a/b) Mainly parallel beta sheets (beta-alpha-beta units) Fold: NAD(P)-binding Rossmann-fold domains - core: 3 layers, a/b/a; parallel beta-sheet of 6 strands Superfamily: NAD(P)-binding Rossmann-fold domains Family: Alcohol dehydrogenase-like, C-terminal domain Protein: Alcohol dehydrogenase Species: Human (Homo sapiens), different isozymes

6. 1.1 Structural Classification C-ter NAD(P)-binding Rossmann β-sheets

7. 1.2 Function ADH ● Oxidoreductases (alcohol:NAD+ oxidoreductase) ● Acting on the CH-OH group of donors ● With NAD+ or NADP+ as acceptor

8. 1.2 Function ADH Alcohol + NAD+ Aldehyde + NADH + H+

9. 1.2 Function ADH Alcohol + NAD+ Ketone + NADH + H+

10. 1.3 Metabolic Pathways involved ● Glycolysis / Gluconeogenesis ● Fatty acid metabolism ● Bile acid biosynthesis ● Tyrosine metabolism ● Glycerolipid metabolism ● Methylnaphthalene degradation ● Metabolism of xenobiotics by cytochrome P450 Important enzyme 3 OMIM Diseases characterized

11. 1.4 ADH system ADH system in vertebrates. Superfamily Family Subfamily Class Coenzyme MDR ADH FADH ADH3 NAD(H) ADH animals ADH1 NAD(H) ADH2 NAD(H) ADH4 NAD(H) ADH5 NAD(H) ADH7 NAD(H) ADH8 NADP(H) Different Isoenzymes

12. 1.4 ADH system ● Isoenzymes generated evolutively by genic duplication --> same ancestor ● Differences: – Substrat specificity – Chinetics – Sensibility to the pirazole inhibitor – Sequence identity ● Intra-species = 50-70 % ● Inter-species = 70-90%

13. ADH Class 1 ● Mainly liver distribution (1% liver proteins) ● High inhibition by pirazole and derivates ● Substrats: – Primary and Secondary aliphatic alcohols – Cyclic and aromathycs alcohols – Biliar acids – Intermediary compounds of dopamine, noradrenaline and serotonine metabolism Liver metabolism

14. ADH Class 1 ● Human ADH-1 codified by 3 genes: – ADH1A – ADH1B – ADH1C ● Subunits α, β, and γ in homodimeric conformation ● β and γ subunits have alelomorfism: different homodimers with only one mutation Arg47His and Arg369Cys Sequence identity of 93-97 % Different chinetics => Dissociation velocity +Kcat with β2β2and β3β3 (Ethanol)

15. ADH Class I ● Isoforms alpha, beta, gamma. ● Beta has three functional polymorphisms. ● Gamma has two.

17. ● The overall sequence similarity is 93%. ● The catalytic site similarity ranges between 50% and 70%.

22. Reaction mechanism ● Zn dependent reaction. ● NAD acts as cofactor. ● The second Zinc atom only has an structural role.

23. The reaction

25. ● The reaction mechanism is the same for the three isoforms. ● Only the topology each protein determines the substrate specificity. ● The alpha isoform has a bigger pocket, and can oxidize secondary alcohols.

27. Evolution of Alcohol DH ● Isozymes in human ● Different species – NAD-binding fold – GroEs-like ● Different protein in the same alcohol DH like family ● Different protein in the same NAD-binding superfamily ● Different protein with the dehydrogenase function

28. Conservation between isozymes

29. Superposition

31. Active Site

32. Conservation between species ● Human ● Horse ● Mouse ● Frog ● Cod

33. NAD-binding Fold

34. Active Site

35. Alignment GroEs-Like Domain

36. GroEs-Like Domain

37. More species ● Pseudomonas ● Bacillus

38. All protein

39. Family Alcohol like Domain ● Alcohol Dehydrogenase ● KetoseReductase ● Quinone Oxidoreductase

42. Superfamily NAD-binding Fold Glutamato DH

44. GroEs-Like Family Sorbitol DH Alcohol DH