Family 6 carbohydrate binding modules in beta-agarases display exquisite selectivity for the non-reducing termini of agarose chains.
Carbohydrate recognition is central to the biological and industrial exploitation of plant structural polysaccharides. These insoluble polymers are recalcitrant to microbial degradation, and enzymes that catalyze this process generally contain non-catalytic carbohydrate binding modules (CBMs) that potentiate activity by increasing substrate binding. Agarose, a repeat of the disaccharide 3,6-anhydro-alpha-L-galactose-(1,3)-beta-D-galactopyranose-(1,4), is the dominant matrix polysaccharide in marine algae, yet the role of CBMs in the hydrolysis of this important polymer has not previously been explored. Here we show that family 6 CBMs, present in two different beta-agarases, bind specifically to the non-reducing end of agarose chains, recognizing only the first repeat of the disaccharide. The crystal structure of one of these modules Aga16B-CBM6-2, in complex with neoagarohexaose, reveals the mechanism by which the protein displays exquisite specificity, targeting the equatorial O4 and the axial O3 of the anhydro-L-galactose. Targeting of the CBM6 to the non-reducing end of agarose chains may direct the appended catalytic modules to areas of the plant cell wall attacked by beta-agarases where the matrix polysaccharide is likely to be more amenable to further enzymic hydrolysis.
Henshaw
Joanna
J
Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
Horne-Bitschy
Ami
A
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada.
van Bueren
Alicia Lammerts
AL
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada.
Money
Victoria A
VA
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom.
Bolam
David N
DN
Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
Czjzek
Mirjam
M
Station Biologique de Roscoff, Vegetaux Marins et Biomolecules, UMR7139-CNRS-UPMC, Place George Teissier, B. P. 74, 29682 Roscoff, France.
Ekborg
Nathan A
NA
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742.
Weiner
Ronald M
RM
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742.
Hutcheson
Steven W
SW
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742.
Davies
Gideon J
GJ
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom.
Boraston
Alisdair B
AB
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada.
Gilbert
Harry J
HJ
Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Electronic address: H.J.Gilbert@ncl.ac.uk.
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D021382:Protein Sorting Signals; D012685:Sepharose / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D002241:Carbohydrates; D010940:Plant Proteins; D011108:Polymers; D011134:Polysaccharides; D021382:Protein Sorting Signals; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase