Genome mining and annotation validation Georges Cohen Institut Pasteur Paris

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

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

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

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

- 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 Lysine fermentation

Lysine fermentation in Fusobacterium nucleatum Lysine 2,3- aminomutase Not sequenced  -Lysine 5,6- aminomutase Not sequenced Not sequenced Acyl-CoA dehydrogenase Butyrate-CoA acetoacetyl-CoA transférase Acetyl-CoA acetyltransférase COOH NH 2 H 2 N COOHH 2 N NH 2 COOH NH 2 NH 2 Lysine kamA kamD,E AtoA,D

Data mining for the 3,5-diaminohexanoate dehydrogenase encoding gene NH 3 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 H2OH2O + NADH + H + NAD + Best candidate: FN1867

Substrate  L-erythro-3,5-diaminohexanoate * * 2 stereoisomeric centers  4 stereoisomers L-erythroD-erythroL-threoD-threo

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

Lysine fermentation in Fusobacterium nucleatum Lysine 2,3- aminomutase Not sequenced  -Lysine 5,6- aminomutase Not sequenced Not sequenced Acyl-CoA dehydrogenase Butyrate-CoA acetoacetyl-CoA transférase Acetyl-CoA acetyltransférase COOH NH 2 H 2 N COOHH 2 N NH 2 COOH NH 2 NH 2 Lysine kamA kamD,E AtoA,D

Enzymatic assay for FN 1868 COOH NH 2 NH 2 COOH ONH 2 + NADNH H2OH2O + + NADHH+H+ 1) Let the product of FN1867 accumulate FN1867 L-erythro-3,5-DAH 3-Keto-5-aminohexanoate SCoA O NH 2 OH OO COOH ONH 2 + acetyl-CoA + FN1868 2) Add then FN1868 and the co-substrate acetyl CoA 3-Keto-5-aminohexanoate 3-aminobutyryl-CoA 3) Follow the disappearance of’acetyl CoA using citrate synthase(CS) acetyl-CoA + oxaloacetate+ DTNB citrate + CoA-disulfite + thionitrobenzoate CS absorbance at 412 nm

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

Annett Kreimeyer Alain Perret Claudine Médigue Marcel Salanoubat Jean Weissenbach J.Biol.Chem.,(2007)282, Georges Cohen, consultant