A Structural and Functional Analysis of α-Glucan Recognition by Family 25 and 26 Carbohydrate-binding Modules Reveals a Conserved Mode of Starch Recognition

Abstract

Starch-hydrolyzing enzymes lacking ␣-glucan-specific carbohydrate-binding modules (CBMs) typically have lowered activity on granular starch relative to their counterparts with CBMs. Thus, consideration of starch recognition by CBMs is a key factor in understanding granular starch hydrolysis. To this end, we have dissected the modular structure of the maltohexaose-forming amylase from Bacillus halodurans (C-125). This five-module protein comprises an N-terminal family 13 catalytic module followed in order by two modules of unknown function, a family 26 CBM (BhCBM26), and a family 25 CBM (BhCBM25). Here we present a comprehensive structure-function analysis of starch and ␣-glucooligosaccharide recognition by BhCBM25 and BhCBM26 using UV methods, isothermal titration calorimetry, and x-ray crystallography. The results reveal that the two CBMs bind ␣-glucooligosaccharides, particularly those containing ␣-1,6 linkages, with different affinities but have similar abilities to bind granular starch. Notably, these CBMs appear to recognize the same binding sites in granular starch. The enhanced affinity of the tandem CBMs for granular starch is suggested to be the main biological advantage for this enzyme to contain two CBMs. Structural studies of the native and ligandbound forms of BhCBM25 and BhCBM26 show a structurally conserved mode of ligand recognition but through non-sequence-conserved residues. Comparison of these CBM structures with other starch-specific CBM structures reveals a generally conserved mode of starch recognition.It is well established that carbohydrates play vital roles in numerous biological settings. They can act as the carriers of biological information, such as in cell development, carcinogenesis, immune response, and cell trafficking; as structural macromolecules, such as the cellulose of plant cell walls or chitin of insect exoskeletons; or as an energy source. Polysaccharides, which are a highly polymerized class of carbohydrates, can perform any of these functions. As mentioned, cellulose and chitin, the first and second most abundant biopolymers on earth, respectively, are the premier structural carbohydrates. Glycosaminoglycans are a highly complex class of polysaccharides that make up the extracellular glue of mammalian tissues and function both in structural and information content roles. Glycogen and starch are related polysaccharides that function as the primary storage carbohydrates in animals and plants, respectively.Glycogen is a polymer of glucose comprising linear ␣-1,4-linked glucose with ␣-1,6 branch points occurring approximately every 8 -12 glucose residues. Amylopectin, a component of starch, is a glycogen-like molecule but with ␣-1,6 branch points occurring approximately every 24 -30 glucose residues. Amylose, the other component of starch, is a polymer of pure linear ␣-1,4-linked glucose. The ␣-1,4 linkages in these polysaccharides make them fold into tight helical structures, resulting in dense granules that function as highly effective storage systems. The release of small...

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…These are widely used in studies of heterogeneous catalytic degradation of insoluble substrates as well as in applications taking advantage of the CBM affinity to direct enzyme-CBM fusion proteins to the surface of the substrate in question (Juge et al 2006). In addition to the first identified SBD (CBM20), 7 CBM families of SBDs have been reported (Boraston et al 2004(Boraston et al , 2006Machovic & Janecek 2006. Moreover, certain regions of the polypeptide chain in GH31 from plants were demonstrated to confer affinity for granular starch (Nakai et al 2008).…”

“…Some common principles may be used to describe the mechanistic action of glycoside hydrolases for degradation of recalcitrant substrates, such as cellulose, other cell wall polysaccharides, or starches (Boraston et al 2004(Boraston et al , 2006Nakai et al 2008). A variety of CBMs representing an array of polysaccharide specificities have been identified (http://www.cazy.org/).…”

A Novel Glucanotransferase that Produces a Cyclomaltopentaose Cyclized by an .ALPHA.-1,6-Linkage

“…In addition, the affinity between the enzyme and the substrate was enhanced by having tandem CBMs. 24) IGTase that has the tandem CBM25 should strongly adsorb raw starch.…”

Determination and Analysis of the Starch Binding Domain of Streptococcus bovis 148 Raw-Starch-Hydrolyzing .ALPHA.-Amylase

“…8,9) More importantly, among CBM family 26 AmyAs, L. amylovorus AmyA and Bacillus halodurans maltohexaose forming amylase (G6 amylase) have been demonstrated to have SBDs. 10) Mizokami 2) and Satoh et al 3) reported that S. bovis 148 AmyA has an ability to adsorb onto raw starch, but they did not analyze its SBD. Few studies on bacterial amylase family s SBD have been reported, especially, expression and functional analysis of SBD itself is rare.…”

“…Few studies on bacterial amylase family s SBD have been reported, especially, expression and functional analysis of SBD itself is rare. 10) In this study, we aim to determine the SBD of S. bovis 148 AmyA and elucidate its role in raw starch degradation.…”