Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B(1)4.
The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixed-linkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixed-linkage β-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.
From the Michael Smith Laboratories and Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
Nicholas
N
McGregor
the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Mariya
M
Morar
From the Michael Smith Laboratories and Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
Thomas Hauch
TH
Fenger
the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Peter
P
Stogios
the Architecture et Fonction des Macromolécules Biologiques, CNRS, Aix-Marseille Université, Marseille 13288, France.
Nicolas
N
Lenfant
From the Michael Smith Laboratories and Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
Victor
V
Yin
the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Xiaohui
X
Xu
the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Elena
E
Evdokimova
the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Hong
H
Cui
the Architecture et Fonction des Macromolécules Biologiques, CNRS, Aix-Marseille Université, Marseille 13288, France, the Department of Biological Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia, and INRA, USC 1408 AFMB, F-13288 Marseille, France.
Bernard
B
Henrissat
the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada, alexei.savchenko@utoronto.ca.
Alexei
A
Savchenko
From the Michael Smith Laboratories and Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada, brumer@msl.ubc.ca.
Harry
H
Brumer
D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D044644:Endo-1,3(4)-beta-Glucanase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D010802:Phylogeny; D018720:Prevotella / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
D001051:Apoenzymes; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005936:Glucans; D011994:Recombinant Proteins; D014990:Xylans; C029353:xyloglucan; D002482:Cellulose; D006026:Glycoside Hydrolases; C407024:endoglucanase Cel5A; C072818:xyloglucan endo(1-4)-beta-D-glucanase; D002480:Cellulase; D044644:Endo-1,3(4)-beta-Glucanase
Bacteroidetes; affinity labeling; carbohydrate chemistry; enzyme inhibitor; enzyme kinetics; microbiome; plant cell wall; polysaccharide; structural biology; structure-function