Genome mining and annotation validation Georges Cohen Institut Pasteur Paris e-mail:gncohen@pasteur.fr

1. Genome mining and annotation validation Georges Cohen Institut Pasteur Paris e-mail:gncohen@pasteur.fr

2. As many as 40% of all predicted genes in completed prokaryotic genomes have no functional annotation

3. Many genes have a predicted function, but that prediction has not been experimentally validated

4. As many as 5-10% of predicted gene functions may be incorrect

5. Many known enzymes have no corresponding genes identified in the sequence databases

6. Lysine fermentation - Known since the 50’s -1 mole of lysine is degraded to 1 mole of acetate,1 mole of butyrate and 2 moles NH3 Well studied in Clostridium sticklandii, but also present in Porphyromonas gingivalis and Fusobacterium nucleatum

7. Acyl-CoA dehydrogenase Not sequenced Butyrate-CoA acetoacetyl-CoA transférase Acetyl-CoA acetyltransférase Lysine fermentation in Fusobacterium nucleatum Lysine 2,3- aminomutase N H H N C O O H 2 b-Lysine 5,6- aminomutase 2 H N C O O H 2 kamA N H 2 kamD,E L y s i n e N H N H 2 2 C O O H Not sequenced Not sequenced AtoA,D

8. Best candidate: FN1867 Data mining for the 3,5-diaminohexanoate dehydrogenase encoding gene H2O + NADH + H+ NAD + NH3 Characteristics of 3,5-diaminohexanoate dehydrogenase: -isolated and purified from Clostridium SB4,Clostridium sticklandii, Brevibacterium L5 - cofactor: NAD+ - molecular weight between 37 and 39 kDa - dimer or tetramer  Search for a F.nucleatum protein which a) possesses a binding site for NAD+ b) has a molecular weight around 38 kDa

9. * * Substrate  L-erythro-3,5-diaminohexanoate 2 stereoisomeric centers  4 stereoisomers L-erythro D-erythro L-threo D-threo

10. Synthesis of DL-erythro-3,5-diaminohexanoate Références: Chem. Berichte 1904, 37, 2357-2362 Organic Preparations and Procedures Int. 1973, 5, 31-35 + NH3 + HCl 6 h reflux 150 °C, 20 h Under pressure Sorbic acid DL-erythro- 3,5-diaminohexanoate - Separation of erythro and threo by recrystallisation in isopropanol - no separation of the D et L isomers

11. Acyl-CoA dehydrogenase Not sequenced Butyrate-CoA acetoacetyl-CoA transférase Acetyl-CoA acetyltransférase Lysine fermentation in Fusobacterium nucleatum Lysine 2,3- aminomutase N H H N C O O H 2 b-Lysine 5,6- aminomutase 2 H N C O O H 2 kamA N H 2 kamD,E L y s i n e N H N H 2 2 C O O H Not sequenced Not sequenced AtoA,D

12. 1) Let the product of FN1867 accumulate FN1867 N H N H + + + + + 2 2 NAD NH4+ H2O NADH H+ C O O H L-erythro-3,5-DAH 3-Keto-5-aminohexanoate 2) Add then FN1868 and the co-substrate acetyl CoA FN1868 + + acetyl-CoA N H O 2 3-Keto-5-aminohexanoate 3-aminobutyryl-CoA S C o A N N H H O O 2 2 C C O O O O H H O O 3) Follow the disappearance of’acetyl CoA using citrate synthase(CS) O H CS acetyl-CoA + oxaloacetate+ DTNB citrate + CoA-disulfite + thionitrobenzoate absorbance at 412 nm Enzymatic assay for FN 1868

13. Tri-coupled assay for FN1869 FN1867 FN1868 Diaminohexanoate------> 3-keto-5-aminohexanoate----->3-aminobutyryl CoA FN1869 ----- ----> Crotonyl CoA

14. Annett KreimeyerAlain PerretClaudine MédigueMarcel SalanoubatJean WeissenbachJ.Biol.Chem.,(2007)282,7191-7Georges Cohen, consultant