Transcript Yvonne Schmidt

Biosynthetic studies of the
cyclocarbamate SB-253514
DFG Research Unit 854 Post-Genomic Strategies for New Antibiotic Drugs and Targets
Yvonne Schmidt1, Jos Raaijmakers2, Harald Gross1
1University
of Bonn, Institute for Pharmaceutical Biology, Nussallee 6, 53115 Bonn, Germany
2Wageningen University, Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
Introduction
Lipoprotein associated phospholipase A2 (LpPLA2) is responsible for hydrolysis of modified oxidized phospholipids from low density lipoprotein causing
the release of pro-inflammatory lyso-phosphatidyl choline and oxidatively modified fatty acids. Inhibition of LpPLA2 is therefore considered a novel
therapeutic strategy in the treatment of diseases that have an inflammatory component such as atherosclerosis. One of these metabolites is SB-253514,
a glycosylated lipopeptide produced by Pseudomonas fluorescens. The structure consists of a 5,5-bicyclic carbamate moiety, connected via two carbons
and one nitrogen atom to myristic acid, which is in turn glycosylated with rhamnose.
During a genomic-driven screening for lipopeptide gene clusters, we recently came across the corresponding gene cluster of SB-253514 which involves
unexpectedly a two modular NRPS gene cluster. Both, its unique structure and its powerful Lp-PLA2-inhibition prompted our study of its biosynthesis.
Of particular interest is hereby the modification of the putative involved second amino acid aside proline and the formation of the cyclocarbamate
structure.
SB-253514 Gene cluster
GT
NRPS
A
C
T
Serine
MO
C
A
LuxR
ES
T
Te
Fig. 1
Fosmid C52 5-1contig 6 RC 12814 bp
GT: Glycosyltransferase 1 Type B
(Rhamnosyl-transferase)
MO: FAD-dependent Monooxygenase
LuxR: Transcription-regulator LuxR Type
ES: Effluxsystem RND-Type
Proline
1001010101010
10111010010011
1010010100101
Proof of the cluster
Predicted structure
Comparative metabolite Screening by LC-MS
(Knockout vs Wildtype)
HO
XIC of +Q1: Exp 1, 554 to 556 Da from Sample 12 (C 52-WT) of 2010-02-26_1.wiff (Turbo Spray)
N
Max. 1,5e7 cps.
1,4e7
Wildtype
1,3e7
HO
1,2e7
O
O
1,1e7
OH
N
H
22,64
1,5e7
extracted
mass:
O
CH3
n
I n t e n s it y , c p s
1,0e7
554 –
556 m/z
According to bioinformatics, the predicted
structure should be either a lipodipeptide
including proline and serine or would result
in a diketopiperazine moiety bearing a
3-hydroxy fatty acid.
9,0e6
8,0e6
7,0e6
6,0e6
5,0e6
4,0e6
3,0e6
Knockout
2,0e6
Isolated structures
22,02
1,0e6
0,0
2
4
6
8
10
12
14
16
Time, min
18
20
22
24
26
28
30
O
Further Bioactivity
H
N
O
OH
O
O
OH
N
Determination of the MIC (minimal inhibition concentration)
O
SB-253514
Test organisms
Mycobacterium smegmatis
50 µg/ml
Staphylococcus aureus SG 511
25 µg/ml
Staphylococcus aureus SG 511
12,5 µg/ml
Bacillus subtilis
12,5 µg/ml
Bacillus subtilis
Listeria welchimeri
strain number
O
CH3
SB-253514
5185
3 mm
SG 511
3 mm
168
4 mm
DSM 20650
3 mm
O
OH
CH3
Feeding studies
O
H
N
OH
O
O
OH
N
3 mm
Corynebacterium diphtheriae
3 mm
Corynebacterium xerosis
OH
new SB-253514 congener
Inhibition zone
Corynebacterium
pseudodiphtheriticum
Mycobacterium smegmatis
O
OH
Bacterial inhibition assay
Staphylococcus aureus
O
25 µg/ml
Listeria welchimeri
Methicillin-sensitive S. aureus
O
12,5 µg/ml
Bacillus subtilis
Gram positive strains
OH
N
O
O
H
N
ATCC 70084
3 mm
Va 167198
3 mm
O
O
O
OH
CH3
Labeling studies were performed in order
to unravel the biosynthetic mechanism(s)
involved in the massive rearrangement of
the peptidic moiety leading to the
cyclocarbamate skeleton. Feeding of single
and double labeled sodium acetate
provided an indirect proof for proline
incorporation and confirmed the
biosynthetic origin of the fatty acid.