The human gut microbial community influences many aspects of human physiology via the output of short chain fatty acids from the fermentation of dietary carbohydrates. Ruminococcus bromii, a keystone species in the human gut, degrades dietary resistant starch. The byproducts of this degradation cross-feed other gut bacteria that produce butyrate, a short chain fatty acid with potent pro-health properties. However, the molecular determinants of resistant starch degradation that initiate the

more »

... olic cascade leading to increased colonic butyrate are unknown. R. bromii exports a unique suite of starch-active proteins that work in concert within larger complexes, amylosomes, that allow this bacterium to bind and degrade resistant starch. Although many gut bacteria have starch-binding and starch-degrading proteins, few can utilize resistant starch as the sole carbon source. The work presented here addresses how individual starch-active proteins from R. bromii facilitate the recognition of resistant starch, allowing its breakdown. In this thesis, I present a structure-function study of the maltogenic α-amylase, Amy5, and two starch-binding proteins of the amylosome system, Sas20 and Sca5. Amy5 is not predicted to be a part of the amylosome, however, it is secreted into the extracellular space where it can encounter its preferred substrate, amylose. Sas20 has two starch-binding domains, the first with a preference for the non-reducing end of starch glycans and the second for starch glycan chains longer than seven glucose residues with helical character. Sca5 has two cohesin modules which have a typical jelly-roll fold and two starch-binding modules, one of which has high sequence homology to domain 2 of Sas20. We propose two cooperative models to describe how Amy5, Sas20, and Sca5 work together to degrade resistant starch. On the surface of the starch granule, Amy5 may attack amylose and open the starch granule so that Sas20 and Sca5 within the amylosome can bind, bringing its starch-active glycoside hydrolase family 1 [...]