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Carbohydrate Binding Module Family 32
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In 1994, the first structure of a carbohydrate-binding module of family 32, in complex with D-galactose, was determined from a fungal galactose oxidase . Following that, a CBM32 from a multi-modular sialidase produced by Micromonospora viridifaciens was shown to demonstrate galactose and lactose binding specificity [2, 3]. A CBM32 from a Cellvibrio mixtus family 16 glycoside hydrolase binds laminarin and pustulan , while a CBM32 from a Clostridium thermocellum mannanase has demonstrable binding on the non-reducing end of β-mannans and β-1,4-linked mannooligosaccharides . A periplasmic-binding protein, YeCBM32, from Yersinia enterolitica shares sequence identity with the CBM32 family and binds the polygalaturonic acid components of pectin . The Clostridium perfringens CBM32s have been well studied and many of their ligand specificities were determined as follows: D-galactose, N-acetyl-D-galactosamine [7, 8, 9], D-galactose-β-1,4-N-acetyl-D-glucosamine (LacNAc), L-fucose-α-1,2-D-galactose-β-1,4-N-acetyl-D-glucosamine (type II blood group H-trisaccharide) , N-acetyl-D-glucosamine, N-acetyl-D-glucosamine-β-1,3-N-acetyl-D-galactosamine, N-acetyl-D-glucosamine-β-1,2-D-mannose, N-acetyl-D-glucosamine-β-1,3-D-mannose (non-biological) , and N-acetyl-D-glucosamine-α-1,4-D-galactose . Some members of CBM family 32 have exhibited a degree of substrate promiscuity; these include CpCBM32-2 from the NagH enzyme and CpCBM32C from the NagJ enzyme of Clostridium perfringens [9, 10]. This CBM family has a very diverse set of ligand specificities that is reflected in the notable amino acid sequence divergence throughout the family .
The CBM32s share the common beta sandwich fold and have a bound structural metal ion that is most often attributed to be calcium . Most family members have fairly weak binding affinities (Ka values in the mM-1 and low μM-1 range) [5, 9, 10]. These binding site are located at the terminal loop region within the CBM32 family. The binding sites are, in some cases, quite shallow and designed to bind monosaccharides or short oligosaccharides, making these type C CBMs [7, 8, 9, 10]. Variability within the apical loop region within the family confers the different ligand specificities. Characteristically, there is one residue, commonly Trp but also Tyr, which provides an important hydrophobic platform for interaction with the ring of one of the sugar moieties (Figure 1) [2, 8, 9]. In most cases, the CBM32 members interact with the non-reducing end of oligosaccharides [5, 7, 8, 9, 10]. However, this is not always the case, as demonstrated by the periplasmic-binding protein from Y. enterocolitica, which shares sequence identity with the CBM32 family  and binds polygalacturonic acid polymers . Some structural examples of the complex oligosaccharide binding sites of the CBM32s can be found in the following PDB entries: 1EUU , 2J7M , 4A45 , 4A6O , and 2W1U , to name just a few. An updated list of available three-dimensional structures is available on the CBM32 page of the CAZy Database.
CBMs, in general, are thought to target pendant catalytic modules to their respective substrates. CtCBM32 from a C. thermocellum manannase potentiates the hydrolytic function of the enzyme and prevents transglycosylation reactions by preferentially binding the non-reducing end of β-mannans. Thus, the CBM32 may sterically block the access of longer manno-oligosaccharide acceptor molecules to the enzyme active site thereby enhancing hydrolysis and affecting the products of catalysis . CBM32s from the Gram positive pathogen C. perfringens may well have a dual role as many of the enzymes containing CBM32s have an LPXTG motif at their C-terminal end, which generally signals for sortase-mediated anchoring to the bacterial cell wall . Thus, not only would the catalytic modules be targeted to substrate, but also the bacterium as a whole, suggesting an adhesin-like activity for these CBMs .
The types of catalytic modules that the CBM32 members are associated with vary widely and include sialidases , β-N-acetylglucosaminidases , α-N-acetylglucosaminidases , mannanases  and galactose oxidases . In enteric bacteria the CBM32 motif may occur more than once in the same enzyme and they may or may not share the same ligand specifities; this suggests the possibility of heterogenic multivalent binding events [7, 8, 11]. Other modules that may be associated in the same enzymes are different families of CBMs, FNIII domains, and cohesin and dockerin domains [8, 15, 18, 19]. There are now examples of CBM32s that are independant of a catalytic module, such as the YeCBM32 periplasmic-binding protein .
The ligand binding loops from CpCBM32-2 from the NagH enzyme were modified to create polyol-responsive antibody mimetics for affinity purification of specific proteins , this is an unusual biotechnological application for a CBM and it speaks to the plasticity within the loop binding region.
- First Identified
- The galactose-binding function of CBM32 was first suggested by crystallography of a galactose oxidase from the fungus Fusarium graminearum  (previously Dactylium dendroides ).
- First Structural Characterization
- In 1991, the first crystal structures of a galactose oxidase from the fungus F. graminearum (e.g. PDB ID 1GOF) revealed an N-terminal CBM32, although this study did not explore the carbohydrate-binding function of this module . The first available complex structure of a CBM32 is from the same galactose oxidase soaked with galactose ; unfortunately, a corresponding entry in the RCSB Protein Data Bank is lacking.
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- Ögel, Z.B. Brayford, D. and McPherson, M.J. (1994) Cellulose-Triggered Sporulation in the Galactose Oxidase-Producing Fungus Cladobotryum (Dactylium) Dendroides Nrrl-2903 and Its Reidentification as a Species of Fusarium. Mycological Research, 98(4):474-480. DOI:10.1016/S0953-7562(09)81207-0
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