Characterization of Arabinosyl Transfer Reactions in the Biosynthesis of Mycobacterial Cell Envelope (Lipo)Polysaccharides

Abstract

D-Arabinofuranose is a major glycosyl constituent of mycobacteria found in two essential cell envelope heteropolysaccharides, arabinogalactan and lipoarabinomannan. Seven different arabinosyltransferases at least are required to synthesize the arabinan domain of these two major glycans. Because of their interest from the perspective of drug development, these enzymes have been the object of intense investigations. In this chapter, we describe the protocols used to perform nonradioactive arabinosyltransferase assays with synthetic acceptor and donor substrates and characterize the enzymatic products of the reactions by mass spectrometry.

Keywords: Arabinosyltransferase; D-Arabinose; Lipid donor; Mycobacteria; Synthetic arabinoside acceptor.

Figure 1.

Arabinosyltransferases involved in the biosynthesis of the arabinan domains of lipoarabinomannan (left panel) and arabinogalactan (right panel) in M. tuberculosis.

Figure 2.. Structures of the synthetic acceptor and donor substrates used in the arabinosyltransferase reactions described in this chapter.

(A) Chemical structure of decaprenylphosphoryl-β-D-arabinose (DPA), the only known arabinose donor in mycobacteria. (B) Structures of synthetic dimannoside (α-D-Manp-(1→6)- α-D-Manp-O(CH₂)7CH₃) and trigalactan (β-D-Galf-(1→5)-β-D-Galf-(1→6)-β-D-Galf-O(CH2)₇CH₃) acceptor substrates. (C) Schematic representation of arabinosyltransferase reactions using M. smegmatis membranes as enzyme source, DPA as the arabinose donor and the synthetic dimannoside and trigalactan acceptors shown in (B).

Figure 3.. LC-MS analysis of permethylated reaction products from in vitro cell-free arabinosyltransferase reactions.

The arabinose acceptors shown in Fig. 2B and their enzymatic products are mostly detected as their ammonium adducts [M+NH₄]⁺ in the positive ion mode. (A–C) Total ion chromatograms. The two major peaks indicated with an arrow correspond to the dimannoside (B) and trigalactan (C) acceptors. These peaks are lacking in the control reaction devoid of acceptor substrate (A). The asterisks indicate their corresponding enzymatic products. Elution profiles (D–G) and extracted ion chromatograms (H–K). The elution profiles of the dimannoside acceptor (m/z 570.38) at 21.9 min (D) and trigalactan acceptor (m/z 770.48) at 22.4 min (E) are shown. Two new peaks with retention times 22.2 min (F) and 22.7 min (G) correspond to the addition of an arabinosyl residue to the dimannoside and trigalactan acceptors, respectively. The presence of ions with m/z 730.45 (J) and 934.55 (K) further confirms the enzymatic transfer of a single arabinosyl residue onto the synthetic dimannoside and trigalactan acceptors, respectively. The extracted ion chromatograms of the two acceptor substrates are shown in panels H and I. The chemical structures of the acceptors and their possible enzymatic products are shown alongside their corresponding extracted ion chromatograms.