How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity Fiona Cuskina,b,1, James E. Flinta,1, Tracey M. Glosterc,1,2, Carl Morlanda, Arnaud Basléa, Bernard Henrissatd, Pedro M. Coutinhod, Andrea Strazzullie, Alexandra S. Solovyovaa, Gideon J. Daviesc, and Harry J. Gilberta,b,3 aInstitute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; bThe Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602; cStructural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom; dArchitecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, 13288 Marseille Cedex 9, France; and eInstitute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, 80131 Naples, Italy Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved October 30, 2012 (received for review July 16, 2012) Noncatalytic carbohydrate binding modules (CBMs) are components CBMs enhance the activity of their cognate enzymes, and though of glycoside hydrolases that attack generally inaccessible substrates. the mechanism(s) by which this occurs remains uncertain, CBMs CBMs mediate a two- to fivefold elevation in the activity of endo- most likely fulfill a targeting function by increasing the effective acting enzymes, likely through increasing the concentration of the concentration of the appended enzymes, in the vicinity of the appended enzymes in the vicinity of the substrate. The function of substrate, thereby enhancing catalytic efficiency (8, 9). CBMs appended to exo-acting glycoside hydrolases is unclear Fructans, such as inulin and levan (polymers of predominantly because their typical endo-binding mode would not fulfill a target- β-2,1– or β-2,6–linked fructose units, respectively) are common ing role. Here we show that the Bacillus subtilis exo-acting β-fruc- dietary plant polysaccharides that are used extensively as pre- fi tosidase SacC, which speci cally hydrolyses levan, contains the biotics, to ensure that the human large-bowel microbiota max- founding member of CBM family 66 (CBM66). The SacC-derived imizes human health by selecting for beneficial bacteria such as CBM66 (BsCBM66) targets the terminal fructosides of the major fi BIOCHEMISTRY Bs Bi dobacter (10). Fructans are metabolized by GH68 and, more fructans found in nature. The crystal structure of CBM66 in com- frequently, by GH32 enzymes that include endo- and exo-acting plex with ligands reveals extensive interactions with the terminal levanases, inulinases, and nonspecific β-fructosidases, whereas fructose moiety (Fru-3) of levantriose but only limited hydrophobic transglycosylases catalyze the synthesis of these fructans (11). contacts with Fru-2, explaining why the CBM displays broad specific- ity. Removal of BsCBM66 from SacC results in a ∼100-fold reduction Understanding the mechanism(s) by which GH32 enzymes display in activity against levan. The truncated enzyme functions as a non- such a range of different activities is important when designing specific β-fructosidase displaying similar activity against β-2,1– and fructan-based prebiotic strategies. β-2,6–linked fructans and their respective fructooligosaccharides. The catalytic modules of GH32 fructanases consist of a domain fi β Conversely, appending BsCBM66 to BT3082, a nonspecific β-fructosi- that adopts a ve-bladed -propeller fold and houses the active site, β dase from Bacteroides thetaiotaomicron, confers exolevanase activ- which is abutted onto a -sandwich domain that likely plays a struc- ityontheenzyme.WeproposethatBsCBM66 confers specificity for tural role (12). A cohort of GH32 enzymes, exemplified by SacC levan, a branched fructan, through an “avidity” mechanism in which from Bacillus subtilis, an exo-acting levanase (13), contain, in addi- the CBM and the catalytic module target the termini of different tion to the catalytic module, a ∼160-residue sequence of unknown branches of the same polysaccharide molecule. This report identifies function. Here we show that the C-terminal 160-residue module a unique mechanism by which CBMs modulate enzyme function, of SacC (designated BsCBM66) is the founding member of CBM and shows how specificity can be tailored by integrating nonspecific family 66 (CBM66) that binds to the nonreducing end of fructan catalytic and binding modules into a single enzyme. polymers. Intriguingly, BsCBM66, despite displaying broad speci- ficity, directs nonspecific β-fructosidases onto highly branched fruc- isothermal titration calorimetry | X-ray crystallography | prebiotics | tans. This study thus provides generic insights into how enzymatic biofuels | lectins specificitycanbeachievedthroughthe recruitment of nonspecific catalytic and noncatalytic carbohydrate binding modules into a omplex carbohydrates represent a major nutrient for nu- single protein. This report therefore provides unique strategies Cmerous microbial ecosystems, exemplified by bacterial and for engineering the specificity of carbohydrate modifying enzymes. fungal communities established in the rumen and large bowel of mammals, where they play an important role in animal nutrition and human health, respectively (1, 2). It is also evident that these Author contributions: F.C., J.E.F., T.M.G., B.H., A.S.S., G.J.D., and H.J.G. designed research; composite structures are of increasing industrial significance, F.C., J.E.F., T.M.G., C.M., A.B., B.H., P.M.C., A.S., and A.S.S. performed research; F.C., J.E.F., T.M.G., C.M., A.B., B.H., P.M.C., A.S.S., G.J.D., and H.J.G. analyzed data; and T.M.G. and particularly in the bioenergy and bioprocessing sectors (3). The H.J.G. wrote the paper. enzymes that catalyze the degradation of these complex carbo- The authors declare no conflict of interest. hydrates, primarily glycoside hydrolases but also polysaccharide This article is a PNAS Direct Submission. lyases, are grouped into sequence-based families on the contin- Freely available online through the PNAS open access option. uously updated CAZy database (4). Members of the same family Data deposition: The crystallography, atomic coordinates, and structure factors have been display a common fold, and the catalytic mechanism and catalytic deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4AZZ, 4B1L, and 4B1M). fi apparatus are also conserved (5). Substrate speci city within 1F.C., J.E.F., and T.M.G. contributed equally to this work. glycoside hydrolase families (GHs), however, can vary signif- 2Present address: Biomedical Sciences Research Complex, University of St. Andrews, St. icantly (6). Glycanases that attack inaccessible substrates often Andrews KY16 9ST, United Kingdom. contain noncatalytic carbohydrate binding modules (CBMs; see 3To whom correspondence should be addressed. E-mail: [email protected]. ref. 7 for review) that are also grouped into sequence-based This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. families in the CAZy database. Against recalcitrant substrates, 1073/pnas.1212034109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1212034109 PNAS | December 18, 2012 | vol. 109 | no. 51 | 20889–20894 Downloaded by guest on September 26, 2021 Results oligosaccharides, exhibiting maximal activity against the dis- C-Terminal Domain of the Exolevanase SacC Is the Founding Member accharides of the two fructans. Thus, BsCBM66 does not con- of a Large CBM Family. The B. subtilis exolevanase SacC comprises tribute to the structural integrity of the active site of the enzyme, a GH32 catalytic module and a C-terminal region of unknown which appears to contain only two fructose-binding sites. Although function (14). To test the hypothesis that the C-terminal domain the activities of BsLev32CM and SacC were similar for inulin ∼ of SacC is a CBM, the capacity of the protein module to bind to and several fructooligosaccharides, BsLev32CM was 100-fold complex carbohydrates was evaluated. The data (Table S1) showed less active than SacC against B. subtilis levan, reflecting a ∼10-fold that the C-terminal module binds to levan, but not to any of the increase in Km and a similar decrease in kcat (Table 1). These fi other polysaccharides evaluated, demonstrating that SacC contains data indicate that BsCBM66 is able to target the nonspeci c β β a C-terminal CBM, designated hereafter as BsCBM66. Analysis -fructosidase, BsLev32CM, to levan, a -2,6-linked branched of sequence-based relatives of BsCBM66 using BLAST identified polysaccharide. To demonstrate that the truncation per se has not fl fi ∼140 bacterial protein modules that display significant similarity in uenced enzyme speci city, the activity of a variant of SacC − to the Bacillus CBM (e <10 6 and sequence identity >30%), and (W640A), in which BsCBM66 was inactive, was evaluated. The fl therefore, BsCBM66 is the founding member of CBM66. Notably, W640A mutation did not in uence the activity of SacC against 106 of the CBM66 members are located in GH32 enzymes (Fig. 1; oligosaccharides or inulin, but caused a substantial reduction in Fig. S1), strongly suggesting that the predominant role of CBM66 the capacity of the enzyme to hydrolyze levan (Table 1). This lower is in binding fructans. The remaining CBM66 members are linked activity was similar to that seen for the isolated catalytic domain fi to a range of CAZy enzymes, primarily glycoside hydrolases and BsLev32CM, con rming that a functional BsCBM66 is integral to lyases, associated with plant cell-wall degradation. the elevated activity displayed by SacC against levan. Bs Influence of BsCBM66 on SacC Function. Biochemical analysis revealed CBM66 Targets the Nonreducing End of Fructans. To explore the fi that SacC displays ∼100-fold-higher activity for levan (β-2,6– speci city of BsCBM66 in more detail, isothermal titration calo- fi glycosidic linkages) compared with inulin (β-2,1–glycosidic link- rimetry was used to determine the af nity of the protein for a ages) or oligosaccharides [with a degree of polymerization (DP) range of fructose-containing oligosaccharides and polysaccharides <6] of either fructan (Table 1).