Abstract
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 language | English |
|---|---|
| Article number | e01058-15 |
| Journal | mBio |
| Volume | 6 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 29 Sep 2015 |
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Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii. / Ze, Xiaolei; David, Ben; Laverde Gomez, Jenny; Dassa, Bareket; Sheridan, Paul O; Duncan, Sylvia H; Louis, Petra Gisela Helen; Henrissat, Bernard; Juge, Natahlie ; Koropatkin, Nicole M; Bayer, Edward A.; Flint, Harry J.
In: mBio, Vol. 6, No. 5, e01058-15, 29.09.2015.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii
AU - Ze, Xiaolei
AU - David, Ben
AU - Laverde Gomez, Jenny
AU - Dassa, Bareket
AU - Sheridan, Paul O
AU - Duncan, Sylvia H
AU - Louis, Petra Gisela Helen
AU - Henrissat, Bernard
AU - Juge, Natahlie
AU - Koropatkin, Nicole M
AU - Bayer, Edward A.
AU - Flint, Harry J
N1 - ACKNOWLEDGMENTS We acknowledge support from BBSRC grant no. BB/L009951/1, from the Scottish government Food, Land and People program, and from the Society for Applied Microbiology. E.A.B. is supported by a grant (no. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel, and by a grant from the United States-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of the Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry. Thanks are due to Fergus Nicol for proteomic analysis and to Auriane Bernard for enzyme assays on stationary-phase cultures. We also thank Julian Parkhill and Keith Turner (Wellcome Trust Sanger Institute, Cambridge, United Kingdom) for making the R. bromii L2-63 genome sequence available for analysis.
PY - 2015/9/29
Y1 - 2015/9/29
N2 - Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energyfrom 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 fructoseas an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptideswere detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted tocarry dockerin modules, with 1, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediatethe formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerinsfrom R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymesAmy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wallanchoringprotein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylasesAmy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, asits dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides thefirst example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.”
AB - Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energyfrom 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 fructoseas an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptideswere detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted tocarry dockerin modules, with 1, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediatethe formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerinsfrom R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymesAmy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wallanchoringprotein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylasesAmy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, asits dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides thefirst example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.”
U2 - 10.1128/mBio.01058-15
DO - 10.1128/mBio.01058-15
M3 - Article
VL - 6
JO - mBio
JF - mBio
SN - 2161-2129
IS - 5
M1 - e01058-15
ER -