The singular Corynebacterium glutamicum Emb arabinofuranosyltransferase polymerises the α(1 → 5) arabinan backbone in the early stages of cell wall arabinan biosynthesis

The arabinan-containing polysaccharides, arabinogalactan (AG) and lipoarabinomannan (LAM), are key cell wall components of the Corynebacterineae, which include Corynebacteria, Norcadia and Mycobacteria. Both AG and LAM contain elaborate arabinan domains composed of distinct structural motifs. Mycobacterial EmbA, EmbB and EmbC, collectively known as the Emb proteins, have been identified as arabinosyltransferases (ArafTs), which are targeted by the front-line anti-tubercular drug ethambutol. Previous studies have established that EmbA and EmbB play a role in the synthesis of the characteristic terminal hexa-arabinosuranosyl motif, whilst EmbC is involved exclusively in the biosynthesis of LAM. Herein, we have investigated the role of the singular Emb protein from Corynebacterium glutamicum through the detailed biochemical and chemical analysis of a double ΔaftAΔemb mutant, where the priming Cg-AftA protein, which generates the substrate for Cg-Emb has been deleted. Analysis of its cell wall revealed a complete absence of arabinose resulting in a truncated cell wall containing only a galactan backbone accompanied with complete loss of cell wall bound mycolates. In vitro cell-free assays using C. glutamicumΔaftA, C. glutamicumΔemb, C. glutamicumΔaftAΔemb and C. glutamicumΔaftBΔaftD and two synthetic acceptors, which mimick the arabinofuranose (Araf) “primed” galactan chain, demonstrated that Cg-Emb is able to transfer an Araf residue to the C5 of the Araf positioned on the synthetic acceptor(s). These results indicate that Cg-Emb acts as an α(1 → 5) ArafT and elongates the arabinan core during the early stages of arabinan biosynthesis in C. glutamicum.