Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the d-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the d-arabinan or d-galactan components of arabinogalactan. Using one of these isolates with exo-d-galactofuranosidase activity, we generated enriched d-arabinan and used it to identify a strain of Dysgonomonas gadei as a d-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave d-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-d-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-d-arabinanases with different preferences for the d-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.

Division and degradation of bacterial cell walls requires coordinated action from a myriad of enzymes. This particularly applies to the elaborate cell walls of acid-fast organisms such as Mycobacterium tuberculosis, which consist of a multi-layered cell wall that contains an unusual glycan called arabinogalactan. Enzymes that cleave the D-arabinan core of this structure have not previously been identified in any organism. We have exploited the breadth of carbohydrate active enzymes in the human gut microbiota to identify four families of glycoside hydrolases each with the capability to degrade the D-arabinan or D-galactan components of arabinogalactan. We have discovered novel exo-D-galactofuranosidases from gut bacteria and used them to discover both endo- and exo- acting enzymes that cleave D-arabinan. This includes new members of the DUF2961 family (GH172), and a novel family of glycoside hydrolases (DUF4185) which display endo-ᴅ-arabinofuranase activity. The DUF4185 enzmyes are conserved in mycobacteria and found in many microbes, suggesting that the ability to degrade mycobacterial glycans plays an important role in the biology of diverse organisms. All mycobacteria encode two conserved endo-D-arabinanases that display different preferences for the essential cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important to cell wall modification and/or degradation. Identification of these enzymes will enable isolation and analysis of mycobacterial cell wall components and facilitate the discovery of new therapeutic or diagnostic options for mycobacterial diseases.