Abstract
The microbial enzymes that depolymerize plant cell wall polysaccharides, ultimately promoting energy liberation and carbon recycling, are typically complex in their modularity and often contain carbohydrate-binding modules (CBMs). Here, through analysis of an unknown module from a Thermotoga maritima endo-β-1,4-galactanase, we identify a new family of CBMs that are most frequently found appended to proteins with β-1,4-galactanase activity. Polysaccharide microarray screening, immunofluorescence microscopy, and biochemical analysis of the isolated module demonstrate the specificity of the module, here called TmCBM61, for β-1,4-linked galactose-containing ligands, making it the founding member of family CBM61. The ultra-high resolution X-ray crystal structures of TmCBM61 (0.95 and 1.4 Å resolution) in complex with β-1,4-galactotriose reveal the molecular basis of the specificity of the CBM for β-1,4-galactan. Analysis of these structures provides insight into the recognition of an unexpected helical galactan conformation through a mode of binding that resembles the recognition of starch.
Highlights
These enzymes often contain, in addition to their catalytic module, a carbohydrate-binding module (CBM)2 and sometimes other ancillary modules [1, 2]
Microarrays probed with TmCBM61 were populated with diverse oligo- and polysaccharides derived from plant and algal cell walls
TmCBM61 bound with greatest affinity to pectins and especially to samples containing the -1,4-galactan side chain component of pectin and to -1,4-galactotetraose (Fig. 1, A and G), suggesting specificity for polymers of -1,4-linked galactose
Summary
Cloning—The gene fragments encoding TmCBM61 (amino acids 461– 606 of TM1201, T. maritima GH53 endo--1,4-galactanase [8]) and the TmGH53 catalytic module (amino acids 22–382 of TM1201) were PCR-amplified from T. maritima genomic DNA (strain MS8B, ATCC 43589D-5) using the oligonucleotide primers CBM61-F and CBM61-R for the CBM and GH53-F and GH53-R for the endo--1,4-galactanase catalytic module (see supplemental Table 1 for oligonucleotide sequences). This initial rough model was sufficient to identify the module’s general -sandwich structure, a fold possessed by the majority of CBMs, and allowed the rough placement in the electron density of a polyalanine model based on CBM16-1 from Caldanaerobius polysaccharolyticus (formerly Thermoanaerobacterium polysaccharolyticum; Protein Data Bank code 2ZEX [19]) This polyalanine model was trimmed to its core -sheet structure and was submitted with the gadolinium atoms to PHASER for combined single-wavelength anomalous dispersion/molecular replacement phasing [20]. This improved the electron density maps, allowing a model of ϳ60% completeness to be built At this point in the process, the two high resolution data sets of TmCBM61 in complex with carbohydrate were obtained.
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