Abstract
The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 A resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both beta-manno- and beta-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity.
Highlights
The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance
Type A carbohydrate-binding modules (CBMs) always bind to crystalline cellulose, irrespective of the activity of the parent enzyme, Type B CBMs generally bind to the substrate that is hydrolyzed by the appended catalytic domains
Crystal Structure of CBM29-1—The noncatalytic protein, NCP1, of the P. equi multienzyme complex contains two family 29 CBMs arranged in tandem [34]
Summary
The Escherichia coli strains used in this study were BL21(DE3): pLysS and Origami B:pLysS (Novagen). The plasmids employed in this work were recombinants of the expression vector pET22b (Novagen) encoding CBM29-2 (pVM1), and CBM29-1 (pVM2), which were constructed previously [15, 34]. E. coli was cultured in Luria-Bertoni broth liquid media at 37 °C and 180 rpm unless otherwise stated. The complete sequences of the DNA encoding the CBM29 mutants were determined by MWG-Biotech (Ebersberg, Germany) using T7 forward and reverse primers to confirm that only the desired mutations had been introduced. CBM29-2 was expressed in E. coli BL21(DE3):pLysS as described previously [15, 34]; CBM29-1 was produced in E. coli Origami B:pLysS. CBM29-1 was purified using the same procedure as for CBM29-2 except that the protein was eluted from the Talon resin with 5 mM rather than 100 mM imidazole. The protein concentration was determined from the calculated molar extinction coefficient of CBM29-1 and CBM29-2 at 280 nm, which were 26,860 and 26,150 MϪ1 cmϪ1, respectively, and reduced coefficients of 20,340 and 25,560 MϪ1 cmϪ1 were used for the CBM29-2 tryptophan and tyrosine mutants, respectively
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