Identification of key amino acid residues in <i>Neisseria polysaccharea</i> amylosucrase

Sarçabal, Patricia; Remaud‐Siméon, Magali; Willemot, René‐Marc; Montalk, Gabrielle Potocki de; Svensson, Birte; Monsan, Pierre

doi:10.1016/s0014-5793(00)01567-2

Cited by 45 publications

(47 citation statements)

References 22 publications

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“…Site-directed Mutagenesis-Site-directed mutagenesis of the AS gene was carried out with the QuikChange TM site-directed mutagenesis kit (Stratagene), as previously described (20). The procedure utilized the pGST-AS double-stranded DNA vector and two synthetic oligonucleotide primers, each complementary to opposite strands of the vector.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorimetry-The fluorimetry experiments were performed using the inactive mutant E328Q purified as described previously (20). The spectrum of denaturation was obtained using purified E328Q variant at 50 mg/liter in fluorimetry buffer (30 mM MOPS, 125 mM NaCl, pH 7.0) without any substrate or supplemented with 100 mM sucrose, 20 mM maltoheptaose or 10 g/liter glycogen.…”

Section: Effect Of Sucrose Concentration On the Distribution Of The Pmentioning

confidence: 99%

“…6). It is situated just after His 392 and Asp 393 , which are always conserved in GH family 13 and are known to stabilize the glucosyl-enzyme intermediate (20,23). The role of these four residues (Asp 394 , Arg 446 , Arg 226 , and Arg 415 ) was investigated by site-directed mutagenesis.…”

Section: Molecular Features Possibly Involved In the Polymerizationmentioning

confidence: 99%

“…Unlike glucansucrases of GH family 70 from lactic acid bacteria (dextransucrase, alternansucrase, and mutansucrase) (17), for which a circularly permutated (␤/␣) 8 -barrel-fold is predicted (18), the catalytic A-domain of AS adopts a nonpermutated (␤/␣) 8 -barrel fold like all of the enzymes of GH family 13 (19). Structural analyses supported by single-site mutational experiments enabled the assignment of the nucleophile (Asp 286 ) and of the acid/base catalyst (Glu 328 ) (20). The importance of three additional residues (Asp 393 , His 187 , and His 392 ), also conserved in GH family 13 and known to assist in catalysis, has been demonstrated.…”

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confidence: 99%

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

Albenne

Skov

Mirza

et al. 2004

Journal of Biological Chemistry

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102

114

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Effect Of Sucrose Concentration On the Distribution Of The Pmentioning

confidence: 99%

Section: Molecular Features Possibly Involved In the Polymerizationmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Albenne

Skov

Mirza

et al. 2004

Journal of Biological Chemistry

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102

114

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“…The locations of the four conserved regions (I to IV) in family GH13 (and family GH70) are indicated with dashed boxes. Amylosucrase has a domain loop (BЈ domain; important for polymerizing activity; indicated with a dashed line and circle) consisting of approximately 60 amino acid residues which is located after ␤-strand 7 (182, 183), immediately after the two catalytically important His392 and Asp393 residues located in conserved region IV (165) (Fig. 1).…”

Section: Fig 2 Topology Diagrams Of Members Of ␣-Amylase Family Gh1mentioning

confidence: 99%

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

Hijum

Kralj

Ozimek

et al. 2006

Microbiol Mol Biol Rev

386

315

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SUMMARY Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)8 barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.

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A pH‐Based High‐Throughput Screening of Sucrose‐Utilizing Transglucosidases for the Development of Enzymatic Glucosylation Tools

Champion¹,

Moulis²,

Morel³

et al. 2010

ChemCatChem

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Sucrose‐utilizing transglucosidases are valuable enzymatic tools for the diversification of carbohydrate‐based molecules. Among them, recombinant amylosucrase from Neisseria polysaccharea is a glucansucrase that naturally catalyzes the synthesis of an amylose‐like polymer as well as the transglucosylation of exogenous hydroxylated acceptors. A semirational engineering approach was recently undertaken to redesign the enzyme active site and adapt it to the glucosylation of a nonnatural acceptor, allyl 2‐N‐acetyl‐2‐deoxy‐α‐D‐glucopyranoside (α‐D‐GlcpNAcOAll), to produce a key building block in the chemoenzymatic synthesis of Shigella flexneri 1b serotype O‐antigen repeating unit. This prior work shows the beneficial effect of single amino acid mutations at two positions (228 and 290) on the recognition of the acceptor by amylosucrase. On the basis of these first results, a library of about 8000 amylosucrase variants combining mutations at these two positions is constructed by saturation mutagenesis. The library is prescreened using a novel pH‐sensitive colorimetric screening method for the detection of sucrose‐utilizing amylosucrase variants, thereby reducing by about 95 % the size of the library to be subsequently screened for acceptor glucosylation. Active clones (5 % of the initial library) are then screened for acceptor recognition, leading to the isolation of 20 variants of potential interest for the production of the target disaccharide α‐D‐Glcp‐(1→4)‐α‐D‐GlcpNAc.

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Identification of key amino acid residues in Neisseria polysaccharea amylosucrase

Cited by 45 publications

References 22 publications

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

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

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

A pH‐Based High‐Throughput Screening of Sucrose‐Utilizing Transglucosidases for the Development of Enzymatic Glucosylation Tools

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