Diapositive 1

1

Genome mining and annotation validation Georges
Cohen Institut Pasteur Paris e-mailgncohen_at_paste
ur.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 50s -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
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Lysine fermentation in Fusobacterium nucleatum
Lysine 2,3- aminomutase
N
H
C
O
O
H
H
N
2
b-Lysine 5,6- aminomutase
2
C
O
O
H
H
N
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
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
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Substrate ? L-erythro-3,5-diaminohexanoate
2 stereoisomeric centers ? 4 stereoisomers
L-erythro
D-erythro
L-threo
D-threo
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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
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Lysine fermentation in Fusobacterium nucleatum
Lysine 2,3- aminomutase
N
H
C
O
O
H
H
N
2
b-Lysine 5,6- aminomutase
2
C
O
O
H
H
N
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
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Enzymatic assay for FN 1868
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Tri-coupled assay for FN1869
FN1867
FN1868
Diaminohexanoate------gt 3-keto-5-aminohexanoate---
--gt3-aminobutyryl CoA
FN1869
----gt Crotonyl CoA
-----
14

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