Starch-active enzymes often possess starch-binding domains (SBDs) mediating attachment to starch granules and other high molecular weight sub-strates. SBDs are divided into nine carbohydrate-binding module (CBM) families, and CBM20 is the earliest-assigned and best characterized family. High diversity characterizes CBM20s, which occur in starch-active glyco-side hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch-phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan host-ing most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily relationships. Data on binding to and enzymatic activity towards soluble ligands and starch granules are summarized for wild-type, mutant and chimeric fusion proteins involving CBM20s. Noticeably, whereas CBM20s in amylolytic enzymes confer moderate binding affinities, with dissociation constants in the low micromolar range for the starch mimic b-cyclodextrin, recent findings indicate that CBM20s in regulatory enzymes have weaker, low milli-molar affinities, presumably facilitating dynamic regulation. Structures of CBM20s, including the first example of a full-length glucoamylase featuring both the catalytic domain and the SBD, are summarized, and distinct architectural and functional features of the two SBDs and roles of pivotal amino acids in binding are described. Finally, some applications of SBDs as affinity or immobilization tags and, recently, in biofuel and in planta bioengineering are presented. Abbreviations AMPK, AMP-activated protein kinase; CBM, carbohydrate-binding module; CGTase, cyclodextrin glucanotransferase; DP, degree of polymerization; GA, glucoamylase; GH, glycoside hydrolase; GWD3, glucan, water dikinase 3; ITC, isothermal titration calorimetry; SBD, starch-binding domain; SEX4, starch excess 4 protein.

In the present work enzymatic hydrolysis of arabinoxylan from pretreated corn bran (190 degrees C, 10 min) was evaluated by measuring the release of xylose and arabinose after treatment with a designed minimal mixture of monocomponent enzymes consisting of alpha-L-arabinofuranosidases, an endoxylanase, and a beta-xylosidase. The pretreatment divided the corn bran material approximately 50:50 into soluble and insoluble fractions having A:X ratios of 0.66 and 0.40, respectively. Addition of acetyl xylan esterase to the monocomponent enzyme mixture almost doubled the xylose release from the insoluble substrate fraction and gave release of 1 mol of xylose/mol of acetic acid released, whereas addition of feruloyl esterase promoted release of only approximately 0.4 mol of xylose/mol of ferulic acid released. For the soluble substrate fraction up to 36% of the xylose could be released by the enzymatic treatment. Acetyl xylan esterase addition on top of the minimal monocomponent enzyme mixture resulted in liberation of up to 0.5 mol of xylose/mol of acetic acid released, whereas feruloyl esterase addition released 1 mol of xylose/mol of ferulic acid released from the soluble substrate. The results imply that on the insoluble material the acetyl xylan esterase was more important for the enzymatic degradation than feruloyl esterase, whereas on soluble arabinoxylan the feruloyl esterase seemed to be more important for the release of xylose.

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific ␣-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave ␣-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific ␣-1,2-arabinofuranosidase, CjAbf43A, displays a fivebladed ␤-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for ␣-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

The aim of this work was to investigate the effect on starch molecular and physicochemical properties of down regulation of the R1 protein in potato (Solanum tuberosum L. cv. "Dianella") tubers. Most prominent is a 90% suppression of the phosphate content in the isolated potato tuber starch. The amylopectin chain length distribution profile as determined by HPAEC/PAD was not affected, but the amylose content was increased in the most down-regulated plants. The pasting properties of the transgenic starch revealed a pronounced decrease in peak viscosity and increased setback viscosity as measured using a rapid Visco analyzer. The starch gels displayed an increased hardness and stickiness with a maximum at 1.7 nmol of Glc-6P mg-1 of starch compared to the control lines. At very low phosphate levels (1.4 nmol of Glc-6P mg-1 of starch), the gel hardness was decreased as a result of increased gel brittleness. The increase in gel brittleness is believed to be an effect of an increased proportion of free amylopectin blocklets in the starch as determined by SEC/RI. The possible links between the structural and physicochemical parameters are discussed.

Edited by Michael R. Sussman Keywords:Bioimaging Carbohydrate-binding module 20 Glucan, water dikinase Starch-binding domain Surface plasmon resonance a b s t r a c tThe family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.