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

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

Charlotte Olagnon 1 , Julia Monjaras Feras 2 , Clemens Grünwald-Gruber 3 , Friedrich Altmann 3 , Lukas Kerner 1 , Miguel A. Valvano 2 , Paul Kosma 1

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

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

Breazeale, S. D.; Ribeiro, A. A.; Raetz, C. R. H. J. Biol. Chem. 2003, 278, 24731.

Gronow, S.; Noah, C.; Blumenthal, A.; Lindner, B.; Brade, H. J. Biol. Chem. 2003, 278, 1647.

Tavares-Carreon, F.; Patel, K. B.; Valvano, M. A. Sci. Rep. 2015, 5, 10773.

Hollaus, R.; Kosma, P.; Zamyatina, A. Org. Lett. 2017, 19, 78.

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