Transcript 슬라이드 1

Lacticin 481:
In vitro Reconstitution of
Lantibiotic Synthetase Activity
전영진
Synthesis of
lacticin 481
from LctA.
It requires
the action
of LctM.
New peaks appear in the mass analysis of the mixture of
His6-LctA and His6-LctM after incubation for a while.
Each peak seems to be showing loss of
water molecules.
(Molecular weight of one: 18
two, three, four...: 36, 54, 72...).
De novo MS/MS sequencing of ubiquitin (8.6 kD).
Of the 197 NH2- and COOH-terminal fragment ions
in one CAD and two ECD spectra of ubiquitin, at least
one mass corresponds to dissociation between each pair
of residues. The two vertical lines between each pair
indicate the cleavage of the -CO-NH- bond for the b, y products
and the -NH-CHR- bond for c, z (-CHR-CO- for a).
7708
-72
7636
1. Weight is decreased by
72 that means 4 water
molecules.
2. Analyses of the b- and ytype fragment ions show
that the 72 daltons is lost
from the C-terminal 20
residues.
Usage of mutants
the leader peptide
the propeptide
His6-LctA(5-51) and His6-LctA(10-51) were fully processed by LctM,
but His6-LctA(25-51) was not processed to dehydrated products.
Amino acids at positions 10 to 24 of the leader peptide are important
for posttranslational modification machinery.
His-LctA(1-38)
b56
b55
Ring between Dhb33/
Dha35 and Cys38
could be formed.
(If the ring forms,
b55 and b56 cannot
appear.)
His-LctA(1-38)C38U
Ring between Dhb33/
Dha35 and Sec38
could be formed.
b56
(If the ring forms,
b55 and b56 cannot
appear.)
One more
dehydration
His-LctA-C49S
Dehydration occurred at Ser49 that is added
newly to give a product with 90 Da loss.
His-LctA(1-38)-C49A
Fragmentations between Asn39 and Met40 and
between Asn41 and Ser42 indicate the absence
of the B-ring.
Conclusions
1. As the procedure for synthesis of lacticin 481 proceeds, four dehydrations
occur.
2. The amino acids at positions 10 to 24 (of the leader peptide) are important
for posttranslational modification machinery.
3. The amino acids at positions 39 to 51 are not important for formation of the
B ring.
4. However, one more dehydration at position 49 precludes the formation of
the B ring and the C ring.
5. Loss of Cys48 precludes the formation of the B ring.