Investigation of 4-amino-4-deoxy-l-arabinose transferases from gram-negative bacteria involved in antibiotic resistance

Session: 
S10.1 Lipopolysaccharides
Code: 
OL10.1.3
Location (hall): 
Glucose
Start/end time: 
Thursday, July 4, 2019 - 17:45 to 18:00
Charlotte
Olagnon

Charlotte Olagnon1, Julia Monjaras Feras2, Clemens Grünwald-Gruber3, Friedrich Altmann3, Lukas Kerner1, Miguel A. Valvano2, Paul Kosma1

1University of Natural Resources and Life Sciences, Vienna, Austria, 2Queen's University Belfast, Belfast, United Kingdom, 3University of Natural Resources and Life Sciences, Vienna, Austria

Modification of bacterial lipopolysaccharide (LPS) by addition of 4-Amino-4-deoxy-L-arabinose (Ara4N) to lipid A and Kdo/Ko inner core units has been implicated as a major cause of antibiotic resistance in Gram-negative bacteria due to the masking of anionic charges.[1-3] 4-Amino-4-deoxy-L-arabinose transferases (ArnT) for which functional and structural information is still scarce, utilize α-Ara4N undecaprenylphosphate as donor substrate. In order to study the enzyme mechanism and explore substrate inhibition, a series of both anomeric forms of Ara4N-phosphodiester linked derivatives have been synthesized. First, TIPDS-protected hemiacetal were converted into a separable mixture of the corresponding αand βH-phosphonates[4], followed by formation of the phosphodiester derivatives in fair yields upon PivCl mediated activation and oxidation. Cleavage of the silyl ether and conversion of the 4-azido into the 4-amino group through a Staudinger reduction gave the target compounds containing linear alkyl and isoprenyl lipid extensions (Fig. 1A). Preliminary activity assays using a crude membrane preparation of ArnT and Kdo2-lipid A (KLA) as acceptor and α-L-Ara4N-P-neryl 4αas donor resulted in a robust in vitro conversion of 40% of the Kdo2-lipid A precursor to mono-substituted Kdo2-lipid A containing a single L-Ara4N unit (Fig. 1B). 

Figure 1. Synthetic L-Ara4N donor substrates (A) and enzymatic transfer of 4αto Kdo2-lipid A (B).

Acknowlegements

Financial support from Austrian Science Fund (FWF, grant P28826-N28).

References: 
  1. Breazeale, S. D.; Ribeiro, A. A.; Raetz, C. R. H. J. Biol. Chem. 2003, 278, 24731.
  2. Gronow, S.; Noah, C.; Blumenthal, A.; Lindner, B.; Brade, H. J. Biol. Chem. 2003, 278, 1647.
  3. Tavares-Carreon, F.; Patel, K. B.; Valvano, M. A. Sci. Rep. 2015, 5, 10773.
  4. Hollaus, R.; Kosma, P.; Zamyatina, A. Org. Lett. 2017, 19, 78.

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