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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.

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Presentation Transcript
slide1

Genome mining and annotation validation

Georges Cohen

Institut Pasteur Paris

e-mail:gncohen@pasteur.fr

slide2

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

slide3

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

slide6

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

slide7

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

slide8

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

slide9

*

*

Substrate  L-erythro-3,5-diaminohexanoate

2 stereoisomeric centers  4 stereoisomers

L-erythro

D-erythro

L-threo

D-threo

slide10

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

slide11

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

slide12

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

tri coupled assay for fn1869
Tri-coupled assay for FN1869

FN1867

FN1868

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

FN1869

-----

----> Crotonyl CoA

slide14

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