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...

Myonecrotic isolates of Clostridium perfringens secrete multimodular sialidases, often termed "large sialidases", that contribute to the virulence of this bacterium. NanJ is the largest of the two secreted sialidases at 1173 amino acids and comprises 6 different modules which are, from the N-terminus, a family 32 carbohydrate binding module (CBM), a family 40 CBM, a family 33 glycoside hydrolase, a module of unknown function, a family 82 "X-module" of unknown function, and a module with amino acid similarity to fibronectin type III domains. The hydrolase activity of clostridial sialidases is quite well documented; however, the functions of their accessory domains are entirely uninvestigated. Here we describe the carbohydrate binding activity of the isolated family 32 CBM (CBM32) and the isolated family 40 CBM (CBM40). CBM32 is shown to bind galactose or N-acetylgalactosamine, while CBM40 is sialic acid specific, though both CBMs appear to bind with very low affinities. The crystal structure of CBM32 was determined at 2.25 A in complex with galactose. This revealed what appears to be a very simple galactose binding site. The crystal structure of CBM40 was determined at 2.20 A in complex with a sialic acid containing molecule that it fortuitously crystallized with, revealing the molecular details of the CBM40-sialic acid interaction. Overall, the results indicate that NanJ contains carbohydrate specific binding modules that likely function to target the enzyme to molecules or cells bearing mixed populations of glycans that terminate in either galactose/N-acetylgalactosamine or sialic acid.

The pyrolysis of 2-methyl-2-nitrosopropane has been studied in a static system over the temperature range 1 40-220'. From a consideration of the products and their distribution with time it is shown that a considerable number of elementary reactions participate in the pyrolysis. The significance of these results for mechanisms of nitric oxide-inhibited pyrolyses is discussed.NITRIC OXIDE has been used as an inhibitor of gaseous free-radical chain reactions for more than thirty years.It has generally been accepted1 that the primary reaction is removal of the free-radicals R by reaction (1) to form a nitroso-alkane R + NO _t RNO(1) There are, however, differences of opinion concerning the fate of the nitroso-alkane. It has been suggested that primary and secondary nitroso-alkanes isomerise to the oxime and some direct studies of the reactions of oximes2-5 have been carried out. Other reactions suggested for nitroso-alkanes include :Apart from one study of the thermal decomposition of nitrosomethane at low pressure in a flow system and an i.r. examination of the same reaction in a static system 7 no direct study of the gas reactions of nitrosoalkanes has been reported although many workers have reported on the direct reaction of free radicals with nitric oxide. It was therefore decided to make a direct study of the pyrolysis of 2-methyl-2-nitrosopropane, the simplest tertiary alkyl-nitroso-compound. Pyrolysis of such a nitroso-alkane cannot involve isomerisation to the oxime and hence might be expected to be simpler than for a primary or secondary nitroso-alkane.

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