Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii

Xiaolei Ze, Ben David, Jenny Laverde Gomez, Bareket Dassa, Paul O Sheridan, Sylvia H Duncan, Petra Gisela Helen Louis, Bernard Henrissat, Natahlie Juge, Nicole M Koropatkin, Edward A. Bayer, Harry J Flint

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Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energy
from dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches.
Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family
(GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructose
as an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptides
were detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted to
carry dockerin modules, with 1, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediate
the formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerins
from R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymes
Amy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wallanchoring
protein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylases
Amy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, as
its dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides the
first example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.”
Original languageEnglish
Article numbere01058-15
Number of pages11
Issue number5
Publication statusPublished - 29 Sep 2015

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