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Transcript
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.