Genome mining and annotation validation

Georges Cohen

Institut Pasteur Paris

e-mail:gncohen@pasteur.fr

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 NH3Well studied in Clostridium sticklandii, but also present in Porphyromonas gingivalis and Fusobacterium nucleatum

Lysine fermentation

Lysine fermentation in Fusobacterium nucleatum Lysine 2,3-

aminomutase

C O O H

ON H2

N H3

Notsequenced

-Lysine 5,6-aminomutase

A c e t y l - C o A

S C o A

ON H2

O H

OO

Not sequenced

S C o A

O

S C o A

O

N H3

Not sequenced

Acyl-CoAdehydrogenase

O H

O

S C o A

OO

Butyrate-CoAacetoacetyl-CoA

transférase

A c e t y l - C o A A c e t a t e

C o A S H

A T P

A D P + P i

C o A S H

Acetyl-CoAacetyltransférase

COOH

NH2

H2N

COOHH2N

NH2

COOH

NH2NH2

Lysine kamAkamD,E

AtoA,D

C O O H

N H2

N H2

C O O H

ON H2

Data mining for the 3,5-diaminohexanoate dehydrogenase encoding gene

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

H2O

+ NADH + H+NAD +

Best candidate: FN1867

Substrate L-erythro-3,5-diaminohexanoate

C O O H

N H2

N H2

**2 stereoisomeric centers 4 stereoisomers

C H2

C H

C H2

H C

C H3

C O O H

H2

N

N H2

C H2

H C

C H2

H C

C H3

N H2

N H2

C O O H

C H2

H C

C H2

C H

C H3

N H2

C O O H

H2

N

C H2

C H

C H2

C H

C H3

C O O H

H2

N

H2

N

L-erythro D-erythro L-threo D-threo

C O O H

N

H

O

N H2

C O O H

N H2

N H2

Synthesis of DL-erythro-3,5-diaminohexanoate

Références: Chem. Berichte 1904, 37, 2357-2362 Organic Preparations and Procedures Int. 1973, 5, 31-35

+ NH3

150 °C, 20 hUnder pressure

+ HCl

6 hrefluxSorbic 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

C O O H

ON H2

N H3

Notsequenced

-Lysine 5,6-aminomutase

A c e t y l - C o A

S C o A

ON H2

O H

OO

Not sequenced

S C o A

O

S C o A

O

N H3

Not sequenced

Acyl-CoAdehydrogenase

O H

O

S C o A

OO

Butyrate-CoAacetoacetyl-CoA

transférase

A c e t y l - C o A A c e t a t e

C o A S H

A T P

A D P + P i

C o A S H

Acetyl-CoAacetyltransférase

COOH

NH2

H2N

COOHH2N

NH2

COOH

NH2NH2

Lysine kamAkamD,E

AtoA,D

Enzymatic assay for FN 1868

COOH

NH2NH2

COOH

ONH2+ NAD NH4

++ + H2O + +NADH H+

1) Let the product of FN1867 accumulate

FN1867

L-erythro-3,5-DAH 3-Keto-5-aminohexanoate

SCoA

ONH2

OH

OO

COOH

ONH2+ 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 + thionitrobenzoateCS

absorbance at 412 nm

Tri-coupled assay for FN1869

Diaminohexanoate------> 3-keto-5-aminohexanoate----->3-aminobutyryl CoA

----> Crotonyl CoA

FN1867 FN1868

-----

FN1869

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

Georges Cohen, consultant