**3. Carbohydrate binding modules**

Many of the enzymes involved in the pathway of polysaccharide biosynthesis present a carbohydrate binding domain in its structure. The first carbohydrate binding domain described was a cellulose-binding domain [45-47]; but later it has been found other modules in related enzymes that bind polysaccharides other than cellulose. These findings compelled to redefine the nomenclature of these domains, and now are called CBM (carbohydrate binding module). To date over 300 putative sequences in more than 50 different species have been identified, and binding domains have been classified into 64 families based on amino acid sequence, the substrate binding specificity and structure [48] (see Carbohydrate-Binding Module Family Server, http://afmb.cnrsmrs. fr/~pedro/CAZY/cbm.html).

 Plant Biotechnology for the Development of Design Starches 497 CBMs have been found in several non-hydrolytic and hydrolytic proteins. Those with hydrolytic activity, such as cellulases, have a complex molecular structure comprised of discrete modules (one catalytic domain and one or more CBMs) that are normally linked by unstructured sequences. The CBMs increase the speed of enzymatic reactions by conducting the catalysis in a close and prolonged physical association with substrates [48] The CBMs present in non-hydrolytic proteins constitute a subunit of the catalytic domain hosts generating cohesive multienzyme complex, which lose enzymatic activity when the CBMs are removed from the structure [48]. Therefore, the CBMs have three general roles with respect to the function of their cognate catalytic modules: (i) a proximity effect, (ii) a targeting function and (iii) a disruptive function [49].

496 The Complex World of Polysaccharides

is in leaf or endosperm (Figure 1).

**Starch Synthases SSI SSIIa (E) SSIIb (L) SSIII SSIV (L)**

**Branching Enzymes BEI BEIIa BEIIb (E)**

> **Isoamylase Isa1 Isa2 (L)**

[10,43].

crucial for the generation of longer, clustered linear segments in the amylopectin molecule that can crystallize and increase the density of the polysaccharide [42]. Plants contain four DBE genes, three of which are classified as isoamylases on the basis of their sequence homologies and substrate specificities, and one pullulanase-type debranching enzyme

While the steps leading to the synthesis of starch are common in most cereals, there are differences in the location and engagement of enzymes, depending on whether the synthesis

**Glc1P**

**ADPGlc - PPase cytosolic form (E) plastidic form**

**ATP**

**PPi**

**Figure 1.** Starch biosynthesis pathway in plants from Glc1P. The scheme indicates the involvement of different isoforms in cereal leaf (L) or endosperm (E). When not specified, the enzymes are dual

**Granular Starch**

ADP

**ADP Amylopectin Amylose**

**Granule Bound Starch Synthase GBSSI (E) GBSSII (L)**

**ADPGlc**

Many of the enzymes involved in the pathway of polysaccharide biosynthesis present a carbohydrate binding domain in its structure. The first carbohydrate binding domain described was a cellulose-binding domain [45-47]; but later it has been found other modules in related enzymes that bind polysaccharides other than cellulose. These findings compelled to redefine the nomenclature of these domains, and now are called CBM (carbohydrate binding module). To date over 300 putative sequences in more than 50 different species have been identified, and binding domains have been classified into 64 families based on amino acid sequence, the substrate binding specificity and structure [48] (see Carbohydrate-Binding Module Family Server, http://afmb.cnrsmrs.

localized in both compartments (adapted from [10] and [44]).

**3. Carbohydrate binding modules** 

fr/~pedro/CAZY/cbm.html).

The SBD (starch binding domain) is usually a distinct sequence-structural module that improves the efficiency of an amylolytic enzyme, improving the binding to starch and its hydrolysis. Because this module was first recognised in amylases and thus revealed to cope with raw starch, it was named the raw (granular) starch binding site [50]. At the present, due to the occurrence of SBDs in a wide spectrum of non-amylolytic enzymes, it has become logical to expect a more variable function of these modules. However, there is little evidence that SBD could bind polysaccharides different to starch, although the ability of pure starch binding and degrading seems to be reserved for microorganisms [50,51].

The CBMs have been clasified in ten families based on sequence comparison: (i) CBM20, such as the C-terminal SBD from *Aspergillus niger* glucoamylase; (ii) CBM21, located at the N-terminal domain in amylase proteins; (iii) CBM25, containing one (i.e. β-amylase from *Bacillus circulans*) or two (i.e. *Bacillus sp*. α-amylase) domains; (iv) CBM26, mainly organized in tandem repeats (i.e. C-terminal domains from *Lactobacillus manihotivorans* α-amylase); (v) CBM34, present in the N-terminal domains of neopullulanase, maltogenic amylase and cyclomaltodextrinase; (vi) CBM41, N-terminal SBD, present mostly in bacterial pullulanases; (vii) CBM45, originating from eukaryotic proteins from the plant kingdom (i.e. N-terminal modules of α-glucan water dikinases and α-amylases); (viii) CBM48, which display glycogen-binding properties (including SBD from the GH13 pullulanase and regulatory modules of mammalian AMP-activated protein kinase); (ix) CBM53, SBD modules from SSIII and (x) CBM58, find in α-amylase/neopullulanase of *Bacteroides thetaiotaomicron* showing maltoheptaose binding [52-54] (http://www.cazy.org). This modules becomes important in breaking down the structure of the substrate due to the presence of two polysaccharide-binding sites [55].

Using bioinformatics techniques several SBDs and several sequences exhibiting similarities to SBDs have also been recognised in enzymes and proteins that are not necessarily amylases such as dual-specific phosphatases. These enzymes should deserve special attention because of their participation in various important physiological processes in plants and mammals. It is worth mentioning that in plants these processes concern starch metabolism, whereas in mammals they participate in the metabolism of glycogen [56,57]. The presence of an SBD motif in protein phosphatases reflects their regulatory function since they are involved in polysaccharide metabolism indirectly via modulation of activity of degradative enzymes (i.e. also amylases), such as isoamylase, β-amylase and disproportionating enzyme [85]. In particular, the initial steps of starch degradation at the granule surface are regulated mainly by phosphorylation [50,51]. Furthermore, a starch biosynthetic enzyme, the starch synthase III (SSIII) from *Arabidopsis thaliana* (AtSSIII) has been reported by our group to have a regulatory role in the synthesis of transient starch [33]. This enzyme contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization followed by three in tandem starch-binding domains (SBD D1, D2 and D3, residues 22-591), which bind to raw starch and its individual components, amylose or amylopectin [53,54,58,59]. The adsorption experiments show that the SBD123 region binds preferentially to amylose, and that the D1 domain is mainly responsible for this selective binding. The D2 domain contains two binding sites including amino acid residues Y394 (binding site 1) and W366 (binding site 2) which act in cooperation with the D1 domain in the binding activity while G335 and W340 have a minor role [54]. It is worth mentioning that our work was the first report on the existence of an SBD in a synthesizing enzyme (AtSSIII) and the first experimental evidence of its starch binding capacity.
