Abstract
Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 A. The protein is a beta-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.
Highlights
Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module
Ligand Binding Studies—To evaluate the binding specificity of CBM from family 6 (CBM6) in more detail, the protein was subjected to affinity gel electrophoresis
Isothermal Titration Calorimetry (ITC) was used to quantify the affinity of CBM6 for xylan, xylooligosaccharides, and cellohexaose
Summary
Refinement statistics Resolution range (Å) Rcryst factor (%) (no. of reflections) Rfree factor (%) (5% of reflections) r.m.s.f deviation from ideal geometry Bond lengths (Å) Bond angles (°) Estimated coordinate error (Å) Average B factor for all atoms (Å). Refinement statistics Resolution range (Å) Rcryst factor (%) B Values in parentheses relate to the outermost resolution shell. E Ano compl.: completeness of anomalous measurements. An N-terminal catalytic module that belongs to glycoside hydrolase family 11, an internal dockerin module [17], and a C-terminal CBM6 that binds primarily to insoluble xylan but can interact with insoluble cellulose [18]. We report the crystal structure of the first representative of CBM family 6, Xyn11A CBM6. This has facilitated the definition of a novel CBM superfamily that encompasses CBM6, CBM4, and CBM22. The location of the ligand-binding site in CBM6 is clearly different than that of CBM4 and CBM22
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