Transcript Diapositive 1 - Institut Pasteur

Genome mining and annotation validation
Georges Cohen
Institut Pasteur Paris
e-mail:[email protected]
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
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
Lysine fermentation in Fusobacterium nucleatum
H 2N
COOH
Lysine 2,3aminomutase
H 2N
b-Lysine 5,6-
COOH
kamA
NH 2
Lysine
NH 2
aminomutase
kamD,E
NH 2
O
COOH
NH3
NH2
Not
sequenced
O
SCoA
Acyl-CoA
dehydrogenase
NH 2
SCoA
Not
sequenced
NH3
Not sequenced
NH2
O
O
O
COOH
O
SCoA
OH
Butyrate-CoA
acetoacetyl-CoA
transférase
AtoA,D
Acetyl-CoA
acetyltransférase
O
O
OH
Acetyl-CoA
O
ATP
Acetyl-CoA
SCoA CoASH
ADP + Pi
Acetate
CoASH
Data mining for the 3,5-diaminohexanoate dehydrogenase encoding gene
NH2
NAD +
H2O
NH2
NH2
O
COOH
COOH
+ NADH + H+
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
Best candidate: FN1867
Substrate  L-erythro-3,5-diaminohexanoate
NH2
*
H2N
*
2 stereoisomeric centers  4 stereoisomers
COOH
COOH
COOH
COOH
COOH
CH2
CH2
CH2
CH2
CH
CH2
H2N
NH2
CH
CH3
L-erythro
HC
NH2
HC
CH2
HC
NH2
CH2
NH2
CH3
D-erythro
H 2N
CH
CH3
L-threo
H 2N
CH
CH2
HC
NH2
CH3
D-threo
Synthesis of DL-erythro-3,5-diaminohexanoate
Références: Chem. Berichte 1904, 37, 2357-2362
Organic Preparations and Procedures Int. 1973, 5, 31-35
NH2
+ NH3
COOH
Sorbic acid
150 °C, 20 h
Under pressure
+ HCl
N
H
O
6 h
reflux
- Separation of erythro and threo by recrystallisation
in isopropanol
- no separation of the D et L isomers
NH2
NH2
COOH
DL-erythro3,5-diaminohexanoate
Lysine fermentation in Fusobacterium nucleatum
H 2N
COOH
Lysine 2,3aminomutase
H 2N
b-Lysine 5,6-
COOH
kamA
NH 2
Lysine
NH 2
aminomutase
kamD,E
NH 2
O
COOH
NH3
NH2
Not
sequenced
O
SCoA
Acyl-CoA
dehydrogenase
NH 2
SCoA
Not
sequenced
NH3
Not sequenced
NH2
O
O
O
COOH
O
SCoA
OH
Butyrate-CoA
acetoacetyl-CoA
transférase
AtoA,D
Acetyl-CoA
acetyltransférase
O
O
OH
Acetyl-CoA
O
ATP
Acetyl-CoA
SCoA CoASH
ADP + Pi
Acetate
CoASH
Enzymatic assay for FN 1868
1)
Let the product of FN1867 accumulate
NH2
NH 2
COOH
+
NAD
FN1867
NH 2
O
COOH
L-erythro-3,5-DAH
+
NH4+
+
H2O
+
NADH
+
H+
3-Keto-5-aminohexanoate
2) Add then FN1868 and the co-substrate acetyl CoA
NH2
O
COOH
+
acetyl-CoA
FN1868
NH2
O
O
+
SCoA
3-aminobutyryl-CoA
3-Keto-5-aminohexanoate
OH
3) Follow the disappearance of’acetyl CoA using citrate synthase(CS)
acetyl-CoA + oxaloacetate+ DTNB
CS
O
citrate + CoA-disulfite + thionitrobenzoate
absorbance at 412 nm
Tri-coupled assay for FN1869
FN1867
FN1868
Diaminohexanoate------> 3-keto-5-aminohexanoate----->3-aminobutyryl CoA
FN1869
--------> Crotonyl CoA
Annett Kreimeyer
Alain Perret
Claudine Médigue
Marcel Salanoubat
Jean Weissenbach
J.Biol.Chem.,(2007)282,7191-7
Georges Cohen, consultant