Unique enzymes help gut bacteria competefor food

The systems and strategies used by gut bacteria to digest dietary fibre in our food varies between different species. Research has shown connections between bacterial composition to both health and different diseases. Thus, basic understanding of how the “good” gut bacteriawork is important, for example how well they compete with other bacteria for nutrients in the gut.

Protective groups complicates degradation of dietary fibre

In the gut, bacteria use enzymes, proteins that catalyse chemical reactions, to break down the complex polysaccharides, i.e. longcarbohydrate chains, in dietary fibre into simple sugars. However, some of these polysaccharides are protected by chemical groups, thathinder enzymatic degradation.

“How gut bacteria handle these protective groups has not been studied in detail. In our study, we have explored how the gut bacterium Dysgonomona's mossii degrades the complex plant polysaccharide xylan. This is an important component in dietary fibre, but the carbohydrate chains are protected by several chemical groups that make them difficult to degrade,” says Johan Larsbrink, Associate Professorof Industrial Biotechnology.

Found three enzymes used to remove protective groups

Dysgonomonas mossii belongs to in the phylum Bacteroidetes, which is a dominant group in the gut microbiota of humans, and they areconsidered "good" bacteria. Previous research has shown that in these species, the genes encoding enzymes for degrading carbohydratechains are often found in large gene clusters in the DNA, so-called polysaccharide utilisation loci (PULs).

“We found three interesting enzymes, carbohydrate esterases, with different properties in a PUL in the bacterium, and we have shown howthey are used to remove protective groups from xylan,” says doctoral student Cathleen Kmezik.

The PUL with the esterase genes also contains several other enzymes which degrade complex xylan chains. The clustering of the studied esterases with these other enzymes indicates that the ability to remove protective groups from carbohydrate chains is important for the bacteria to obtain nutrients.

Solved one enzyme's 3D structure

One of the esterases consists of two fused, catalytic, domains, which is rare. If you compare an enzyme to a pair of scissors that cuts specificchemical bonds, this esterase consists of two pairs of scissors physically connected to each other.

“This enables the esterase to cut different chemical bonds that are situated very close to each other. However, one part of this enzyme wasnot very active on the molecules we tested in our lab experiments, but Scott Mazurkewich, a post-doctoral researcher managed to solve its3D structure by X-ray crystallography. This means that we can see exactly what the enzyme looks like down to a tenth of a nanometre scaleand provides us with a better understanding of what the enzyme is actually doing in the gut,” says Cathleen Kmezik.

Removal of protective groups may be important for survival

The ability to remove protective groups from polysaccharides may be important for survival in the gut, according to the researchers. Moreresearch is needed, though, to determine which niches different bacteria have in terms of what they can eat in the gut − and whether it leadsto increased survival and persistence under certain conditions.

Future studies could allow different species of bacteria to grow simultaneously on different carbohydrates with many or few protective groups and compare who "wins" the battle for nutrition. There is also potential for the enzymes to be used industrially to accelerate the enzymatic degradation of plant biomass in the production of biofuels.

More about the study and the esterases

• Read the article in Journal of Biological Chemistry: A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases
• DmCE1A: enzyme from carbohydrate esterase family 1 (CE1), active on acetyl esters and cleaving coumaryl-like molecules of unknownstructure from plant biomass.
• DmCE1B: enzyme consisting of two fused CE1 domains – DmCE1B_nt and DmCE1_ct, connected through a carbohydrate-bindingmodule. Out of the three enzymes, DmCE1B_nt is the only one with clear activity on feruloyl esters, which can crosslink xylanpolysaccharides, and it was also active on acetyl esters. DmCE1B_ct was only weakly active on acetyl esters. Its 3D structure was solvedtogether with the carbohydrate-binding module. The structure indicates that the enzyme targets larger molecules than those tested inthe lab (see figure).
• DmCE6A: enzyme from carbohydrate esterase family 6 (CE6), with significant activity on acetyl esters, both in model substrates and incomplex biomass. The enzyme was shown to strongly contribute to a faster xylan degradation by enzymes targeting the polysaccharideitself (xylanases).