Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.Cellulose, a major component of plant cell walls and of the biomass of the earth, is a chemically invariant polymer comprising of up to 10,000 ␤-1,4-linked-glucosyl residues. In cell walls cellulose is found in the form of linear insoluble microfibrils that result from self-association of cellulose chains subsequent to synthesis by plasma membrane-located complexes. Biophysical studies have shown that cellulose microfibrils can exist in highly ordered crystalline, semi-ordered para-crystalline, and disordered noncrystalline (amorphous) states (1). Cellulose microfibrils extensively encircle plant cells and are intimately associated with other polysaccharide networks in dense composites that comprise primary and secondary cell walls. The orientation of cellulose microfibrils and their connections with other wall polysaccharide networks are key factors in the generation of cell and organ shapes and mechanical strength during plant growth and development (2).The interactions between cellulose chains both within microfibrils and with matrix polysaccharides within the plant cell wall restrict their accessibility to enzyme attack. To overcome this problem glycoside hydrolas...

The key enzyme responsible for beta-carotene conversion into retinal is beta-carotene 15,15'-monoxygenase (BCMO1). Since it has been reported that the conversion of beta-carotene into vitamin A is highly variable in up to 45% of healthy individuals, we hypothesized that genetic polymorphisms in the BCMO1 gene could contribute to the occurrence of the poor converter phenotype. Here we describe the screening of the total open reading frame of the BCMO1 coding region that led to the identification of two common nonsynonymous single nucleotide polymorphisms (R267S: rs12934922; A379V: rs7501331) with variant allele frequencies of 42 and 24%, respectively. In vitro biochemical characterization of the recombinant 267S + 379V double mutant revealed a reduced catalytic activity of BCMO1 by 57% (P<0.001). Assessment of the responsiveness to a pharmacological dose of beta-carotene in female volunteers confirmed that carriers of both the 379V and 267S + 379V variant alleles had a reduced ability to convert beta-carotene, as indicated through reduced retinyl palmitate:beta-carotene ratios in the triglyceride-rich lipoprotein fraction [-32% (P=0.005) and -69% (P=0.001), respectively] and increased fasting beta-carotene concentrations [+160% (P=0.025) and +240% (P=0.041), respectively]. Our data show that there is genetic variability in beta-carotene metabolism and may provide an explanation for the molecular basis of the poor converter phenotype within the population.

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-␤-D-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or ⌬4,5-anhydrogalaturonic acid (⌬4,5-GalA). ⌬4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.carbohydrate protein binding ͉ enzyme targeting ͉ plant cell wall degradation ͉ protein cell attachment ͉ X-ray crystallography

The enzymatic transfer of activated mannose yields mannosides in glycoconjugates and oligo- and polysaccharides. Yet, despite its biological necessity, the mechanism by which glycosyltransferases recognize mannose and catalyze its transfer to acceptor molecules is poorly understood. Here, we report broad high-throughput screening and kinetic analyses of both natural and synthetic substrates of Rhodothermus marinus mannosylglycerate synthase (MGS), which catalyzes the formation of the stress protectant 2-O-alpha-D-mannosyl glycerate. The sequence of MGS indicates that it is at the cusp of inverting and retaining transferases. The structures of apo MGS and complexes with donor and acceptor molecules, including GDP-mannose, combined with mutagenesis of the binding and catalytic sites, unveil the mannosyl transfer center. Nucleotide specificity is as important in GDP-D-mannose recognition as the nature of the donor sugar.

The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity toward heterogeneous substrates, in which the sequence of sugars is variable, is unclear. An excellent example of such heterogeneity is provided by the plant structural polysaccharide glucomannan, which comprises a backbone of beta-1,4-linked glucose and mannose units. beta-Mannanases, located in glycoside hydrolase (GH) families 5 and 26, hydrolyze glucomannan by cleaving the glycosidic bond of mannosides at the -1 subsite. The mechanism by which these enzymes select for glucose or mannose at distal subsites, which is critical to defining their substrate specificity on heterogeneous polymers, is currently unclear. Here we report the biochemical properties and crystal structures of both a GH5 mannanase and a GH26 mannanase and describe the contributions to substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived from Bacillus agaradhaerens, can accommodate glucose or mannose at both its -2 and +1 subsites, while the GH26 Bacillus subtilis mannanase, BsMan26A, displays tight specificity for mannose at its negative binding sites. The crystal structure of BaMan5A reveals that a polar residue at the -2 subsite can make productive contact with the substrate 2-OH group in either its axial (as in mannose) or its equatorial (as in glucose) configuration, while other distal subsites do not exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is able to hydrolyze glucomannan in which the sequence of glucose and mannose is highly variable. The crystal structure of BsMan26A in light of previous studies on the Cellvibrio japonicus GH26 mannanases CjMan26A and CjMan26C reveals that the tighter mannose recognition at the -2 subsite is mediated by polar interactions with the axial 2-OH group of a (4)C(1) ground state mannoside. Mutagenesis studies showed that variants of CjMan26A, from which these polar residues had been removed, do not distinguish between Man and Glc at the -2 subsite, while one of these residues, Arg 361, confers the elevated activity displayed by the enzyme against mannooligosaccharides. The biological rationale for the variable recognition of Man- and Glc-configured sugars by beta-mannanases is discussed.

Glycoside hydrolases that degrade plant cell walls have complex molecular architectures in which one or more catalytic modules are appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs promote binding to polysaccharides and potentiate enzymic hydrolysis. Although there are diverse sequence-based families of xylan-binding CBMs, these modules, in general, recognize both decorated and unsubstituted forms of the target polysaccharide, and thus the evolutionary rationale for this diversity is unclear. Using immunohistochemistry to interrogate the specificity of six xylan-binding CBMs for their target polysaccharides in cell walls has revealed considerable differences in the recognition of plant materials between these protein modules. Family 2b and 15 CBMs bind to xylan in secondary cell walls in a range of dicotyledon species, whereas family 4, 6, and 22 CBMs display a more limited capability to bind to secondary cell walls. A family 35 CBM, which displays more restricted ligand specificity against purified xylans than the other five protein modules, reveals a highly distinctive binding pattern to plant material including the recognition of primary cell walls of certain dicotyledons, a feature shared with CBM15. Differences in the specificity of the CBMs toward walls of wheat grain and maize coleoptiles were also evident. The variation in CBM specificity for ligands located in plant cell walls provides a biological rationale for the repertoire of structurally distinct xylanbinding CBMs present in nature, and points to the utility of these modules in probing the molecular architecture of cell walls.

Noncatalytic carbohydrate binding modules (CBMs) are components of glycoside hydrolases that attack generally inaccessible substrates. CBMs mediate a two-to fivefold elevation in the activity of endoacting enzymes, likely through increasing the concentration of the appended enzymes in the vicinity of the substrate. The function of CBMs appended to exo-acting glycoside hydrolases is unclear because their typical endo-binding mode would not fulfill a targeting role. Here we show that the Bacillus subtilis exo-acting β-fructosidase SacC, which specifically hydrolyses levan, contains the founding member of CBM family 66 (CBM66). The SacC-derived CBM66 (BsCBM66) targets the terminal fructosides of the major fructans found in nature. The crystal structure of BsCBM66 in complex with ligands reveals extensive interactions with the terminal fructose moiety (Fru-3) of levantriose but only limited hydrophobic contacts with Fru-2, explaining why the CBM displays broad specificity. Removal of BsCBM66 from SacC results in a ∼100-fold reduction in activity against levan. The truncated enzyme functions as a nonspecific β-fructosidase displaying similar activity against β-2,1-and β-2,6-linked fructans and their respective fructooligosaccharides. Conversely, appending BsCBM66 to BT3082, a nonspecific β-fructosidase from Bacteroides thetaiotaomicron, confers exolevanase activity on the enzyme. We propose that BsCBM66 confers specificity for levan, a branched fructan, through an "avidity" mechanism in which the CBM and the catalytic module target the termini of different branches of the same polysaccharide molecule. This report identifies a unique mechanism by which CBMs modulate enzyme function, and shows how specificity can be tailored by integrating nonspecific catalytic and binding modules into a single enzyme.isothermal titration calorimetry | X-ray crystallography | prebiotics | biofuels | lectins C omplex carbohydrates represent a major nutrient for numerous microbial ecosystems, exemplified by bacterial and 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 composite structures are of increasing industrial significance, particularly in the bioenergy and bioprocessing sectors (3). The enzymes that catalyze the degradation of these complex carbohydrates, primarily glycoside hydrolases but also polysaccharide lyases, are grouped into sequence-based families on the continuously updated CAZy database (4). Members of the same family display a common fold, and the catalytic mechanism and catalytic apparatus are also conserved (5). Substrate specificity within glycoside hydrolase families (GHs), however, can vary significantly (6). Glycanases that attack inaccessible substrates often contain noncatalytic carbohydrate binding modules (CBMs; see ref. 7 for review) that are also grouped into sequence-based families in the CAZy database. Against recalcitrant substrates, CBMs enhance the activit...

Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing,” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.