Synthetic Phosphodiester‐Linked 4‐Amino‐4‐deoxy‐l‐arabinose Derivatives Demonstrate that ArnT is an Inverting Aminoarabinosyl Transferase

Abstract

Abstract4‐Amino‐4‐deoxy‐l‐arabinopyranose (Ara4N) residues have been linked to antibiotic resistance due to reduction of the negative charge in the lipid A and core regions of the bacterial lipopolysaccharide (LPS). To study the enzymatic transfer of Ara4N onto lipid A, which is catalysed by the ArnT transferase, we chemically synthesised a series of anomeric phosphodiester‐linked lipid Ara4N derivatives containing linear aliphatic chains as well as E‐ and Z‐configured monoterpene units. Coupling reactions were based on sugar‐derived H‐phosphonates, followed by oxidation and global deprotection. The enzymatic Ara4N transfer was performed in vitro with crude membranes from a deep‐rough mutant from Escherichia coli as acceptor. Product formation was detected by TLC and LC‐ESI‐QTOF mass spectrometry. Out of seven analogues tested, only the α‐neryl derivative was accepted by the Burkholderia cenocepacia ArnT protein, leading to substitution of the Kdo2‐lipid A acceptor and thus affording evidence that ArnT is an inverting glycosyl transferase that requires the Z‐configured double bond next to the anomeric phosphate moiety. This approach provides an easily accessible donor substrate for biochemical studies relating to modifications of bacterial LPS that modulate antibiotic resistance and immune recognition.

Lipopolysaccharide Is a 4-Aminoarabinose Donor to Exogenous Polyisoprenyl Phosphates through the Reverse Reaction of the Enzyme ArnT

“…On the other hand, we also note that the availability of BP glycose substrates is limited and that reverse reactions demonstrated here may be used to generate these substrates in vitro . As noted by Olagnon et al ., new strategies for acquiring and evaluating polyisoprenyl-phosphate intermediates will address the lack of available substrates required for the rational design of inhibitors, as well as limitations associated with clarifying the function of enzymes involved in lipid A modifications with cationic glycoses …”

“…As noted by Olagnon et al, new strategies for acquiring and evaluating polyisoprenyl-phosphate intermediates will address the lack of available substrates required for the rational design of inhibitors, as well as limitations associated with clarifying the function of enzymes involved in lipid A modifications with cationic glycoses. 42 Accordingly, this research directly addresses the deficit of polyisoprenoid substrates by demonstrating the utility of two efficient methods used to obtain both native and tagged BPglycoses. Specifically, we demonstrate the unique advantage of 2CN-BP used to selectively probe the activity of endogenous BP utilizing proteins within membrane fractions of both K-and Bstrains of E. coli.…”

“…43 Similarly, our methods revealed an unestablished activity of ArnT, for which prior characterization of the forward reaction has required the isolation of small quantities of native BP-Ara4N or the use of shorter, synthetically prepared Ara4N-linked isoprenoids. 24,42 Reverse catalysis of PGT enzymes, where UDP-sugar is formed from BPP-sugar in the presence of UMP, has been demonstrated in vitro with various initiating PGT and glycosyltransferase enzymes. 44,45 Research has also been directed at harnessing these reactions to obtain complex NDP-linked sugars, akin to our generation of fluorescent BPlinked Ara4N here.…”

Synthesis of C-glycosyl phosphonate derivatives of 4-amino-4-deoxy-α-ʟ-arabinose

“…We have recently set out to study the substrate specificity of ArnT enzymes in more detail using phosphodiester-linked derivatives in both anomeric configurations and containing short lipid appendages in order to define the minimum structural requirements for Ara4N glycosyltransferase substrates [9]. In parallel, we have started to develop C-phosphonate analogues as these have frequently been exploited as potential inhibitors for glycosyl transferases since the carbon-phosphorus bond is not hydrolyzed in the active site of glycosyl transferases [10][11][12][13].…”

Synthesis of C-glycosyl phosphonate derivatives of 4-amino-4-deoxy-α-L-arabinose

“…4-Amino-4-deoxy-L-arabinose units are activated as the phosphodiester linked undecaprenyl derivative [6] that is then transferred by the action of several Ara4N-transferases (ArnT) [7]. Synthesis of potential inhibitors of the biosynthesis of We have recently set out to study the substrate specificity of ArnT enzymes in more detail using phosphodiester linked derivatives in both anomeric configurations and containing short lipid appendages in order to define the minimum structural requirements for Ara4N glycosyltransferase substrates [9]. In parallel, we have started to develop C-phosphonate analogues as these have frequently been exploited as potential inhibitors for glycosyl transferases, since the carbonphosphorus bond is not hydrolyzed in the active site of glycosyl transferases [10][11][12][13].…”