Molecular Basis of the Amylose-like Polymer Formation Catalyzed by Neisseria polysaccharea Amylosucrase

Abstract

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amyloselike polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with ␣-amylases. Exactly how this enzyme catalyzes the formation of ␣-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg 415 , subsite ؉4) and the correct positioning (Asp 394 and Arg 446 , subsite ؉1) of acceptor molecules. On the other hand, Arg 226 (subsites ؉2/؉3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.Amylosucrase (EC 2.4.1.4) is a glucansucrase belonging to glycoside-hydrolase (GH) 1 family 13 (1, 2). 2 This transglucosidase catalyzes the synthesis of an insoluble amylose-like polymer from sucrose (3), a cheap and easily available agroresource. This is in contrast to starch or glycogen synthases (4), which require nucleotide-activated sugar as a donor. Amylosucrase is thus attractive for the industrial synthesis of amyloselike polymers and for the modification of glucans (in particular to form nondigestible glucans) (5). Remarkably, amylosucrase is the only member of GH family 13 displaying polymerase activity and is clearly unique in this family that mainly contains starch-degrading enzymes. Amylosucrase was first isolated in the culture supernatant of Neisseria perflava (3) and later identified in various Neisseria strains (6, 7). Recently, data mining has revealed the presence of genes encoding putative amylosucrases in the genome of many other organisms such as Deinococcus radiodurans (8), Caulobacter crescentus (9), Xanthomonas campestris, Xanthomonas axonopodis (10), and Pirellula sp. (11). Recombinant amylosucrase from Neisseria polysaccharea (AS) has been the most extensively studied amylosucrase. The gene encoding AS (1) has been cloned, and its product has been purified to homogeneity. Characterization of the reaction products synthesized from sucrose substrate showed that sucrose isomers (turanose and trehalulose), glucose, maltose, and maltotriose were also produced besides the insoluble...

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

“…The A231/F260/F184 residues at the +1/+2 subsites are completely conserved in CGTases but are replaced by other amino acids at the same position in R-amylases (36) ( Table 1). Mutations at these subsites have previously highlighted this acceptor region to be essential in the determination of the reaction specificities of GH clan H enzymes (19,(37)(38)(39)(40)(41)(42). The drastic reduction of cyclization activity by these moderately bulkier substitutes A231T/V, A231V/F260W, may be explained by the inefficiency of the final step of the transglycosylation reaction.…”

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

“…This mechanism, however, appears more and more unlikely for GS enzymes in view of the single covalent intermediate identified (128) and mutational identification of a single catalytic triad (38,99). Moreover, only one active site has been identified in amylosucrase (GH13) (3,183).…”

“…For CGTase, amylosucrase, neopullulanase, acarviosyltransferase, and the branching enzyme of family GH13 (3,13,96,101,102,104,105,206) and GS enzymes of family GH70, these regions have been proven to be important for product formation, giving further insights into the structural relatedness of GS and family GH13 enzymes (see below).…”

“…(146). Amylosucrase is the only enzyme from family GH13 that uses sucrose as a substrate to synthesize ␣-glucan polymers containing ␣-(134) glucosidic linkages (3,182). For family GH13 enzymes, high-resolution three-dimensional structures are available, often in complex with substrates, inhibitors, or products (207).…”

Developing and Engineering Enzymes for Manufacturing Amylose

“…14) Amylosucrase is very attractive since it produces amylose directly from sucrose, a less expensive and renewable biomass. However, the molecular size of amylose produced by this enzyme is reported to be about 10 kDa or smaller.…”